Mutacin I biosynthesis genes and proteins

ABSTRACT

According to the present invention, an isolated and purified DNA sequence which encodes a lantibiotic, mutacin I, is disclosed. The nucleic acid sequence is set forth in SEQ ID No: 1 and the amino acid sequence is set forth in SEQ ID No: 2.

This application is a divisional of application Ser. No. 09/627,376 filed Jul. 28, 2000, now U.S. Pat. No. 6,342,385, published Jan. 29, 2002.

GRANT REFERENCE

The subject invention was made with government support under a grant from the National Institutes of Health (NIH RO 1 DE09082). The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to polypeptide antibiotics and to the identification of genetic loci associated with expression of the antibiotics. The invention particularly describes a purified lanthionine-containing antimicrobial agent, DNA encoding the protein, and methods and compositions for treatments employing the antibiotic.

BACKGROUND OF THE INVENTION

Several species of bacteria inhabit the human oral cavity; among them Streptococcus mutans is considered a major etiologic agent responsible for dental caries. Loesche (1986) Microbiol. Rev. 50:353-380. Previous studies showed a certain percentage of clinical isolates of S. mutans producing antimicrobial substances called mutacins. Caufield et al. (1985) Infect. Immun. 48:51-56; Hamada et al. (1975) Arch. Oral Biol. 20:641-648. Mutacins are active against closely related species as well as a surprisingly wide spectrum of other Gram-positive bacteria. Parrot et al. (1990) Can. J. Microbiol. 36:123-130. The ability to produce mutacins, combined with lactic acid production by S. mutans may contribute to the pathogenesis of these bacteria. Kleinberg, p. 605-624, in W. A. Nolte (ed.), Oral microbiology, The C.V. Mosby Company, St. Louis. Production of mutacins by S. mutans and other oral streptococci may also play a protective role to the host against pathogens such as Group A streptococci and Streptococcus pneumoniae. In this respect, mutacins may serve as antimicrobial agents in the future.

Lantibiotics are lanthionine-containing small peptide antibiotics that are produced by gram-positive bacteria. Jung (ed.), p. 1-34, in G. Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics, ESCOM Sci. Publ., Leiden; Sahl et al. (1995) Eur. J. Biochem. 230:827-853. The lantibiotics are ribosomally synthesized and post-translationally modified. The modification reactions include dehydration of serine and threonine residues and the addition of thiol groups from cycteine residues to the double bound to form lanthionines and β-methyllanthionines, respectively. Some dehydrated serine or threonine may remain as such in the mature lantibiotic molecule.

Based on the secondary structures, Jung assigned lantibiotics into two classes, Type-A (linear) and Type-B (globular). Jung (ed.), p. 1-34, in G. Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics, ESCOM Sci. Publ., Leiden. de Vos et al. ((1995) Molecular Microbiol. 17:427-437) and Sahl and Bierbaum (Sahl et al. (1998) Annu. Rev. Microbiol. 52:41-79) further divided each class into subgroups according to their primary peptide sequences. Thus, subgroup AI contains the nisin-like lantibiotics with nisin, subtilin, epidermin and pep5 as the most thoroughly characterized members. Allgaier et al. (1986) Eur. J. Biochem. 160:9-22; Gross et al. (1968) FEBS Lett. 2:61-64; Gross et al. (1971) J. Am. Chem. Soc. 93:4634-4635; Kaletta et al. (1989) Arch. Microbiol. 152:16-19; Well et al. (1990) Eur. J. Biochem. 194:217-223. Subgroup AII consists of lacticin 481, SA-FF22, salivaricin and variacin. Hynes et al. (1993) Appl. Environ. Microbiol. 59:1969-1971; Piard et al. (1993) J. Biol. Chem. 268:16361-16368; Pridmore et al. (1996) Appl. Environ. Microbiol 62:1799-1802; Ross et al. (1993) Appl. Environ. Microbiol. 59:2014-2021. The genes responsible for the biosynthesis of the lantibiotics are organized in operon-like structures. The biosynthesis locus of all members in the subgroup AI lantibiotics consists of lanA, the structural gene for the lantibiotic; lanB and lanC, the modifying enzyme genes for post-translational modification of the preprolantibiotic; lanP, the protease gene for processing of the prelantibiotic; and lanT, the ABC transporter for secretion of the lantibiotic. In addition, epidermin and gallidermin have an extra gene, lanD, which is responsible for the C-terminal oxidative decarboxylation of the lantibiotic. Kupke et al. (1994) J. Biol. Chem. 269:5653-5659; Kupke et al. (1995) J. Biol. Chem. 270:11282-89. In comparison, subgroup AII lantibiotics have simpler genomic organizations. In subgroup AII, lanB and lanC are combined into one gene, lanM, and lanP and lanT are combined into lanT. Chen et al. (1999) Appl. Environ. Microbiol. 65:1356-1360; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658; Rince et al. (1994) Appl. Environ. Microbiol. 60:1652-1657. All lantibiotic loci also contain a set of immunity genes, which are responsible for self-protection of the producer strains. Saris et al. (1996) Antonie van Leewenhoek 69:151-159. Moreover, the expression of the lantibiotic genes is usually regulated either by a single transcription regulator (Peschel et al. (1993) Mol. Microbiol. 9:31-39; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658) or by a two-component signal transduction system (de Ruyter et al. (1996) J. Bacteriol. 178:3434-3439; Klein et al. (1993) Appl. Environ. Microbiol. 59:296-303; Kuipers et al. (1995) J. Biol. Chem. 270:27295-27304).

Previously, the isolation, biochemical and genetic characterizations of mutacin II, produced by a group II strain of the oral bacteria Streptococcus mutans was reported. Chen et al. (1999) Appl. Environ. Microbiol 65:1356-1360; Novak et al. (1994) J. Bacteriol. 176:4316-4320; Novak et al. (1996) Anal Biochem. 236:358-360; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658. Mutacin II belongs to subgroup AII in the lantibiotic family. Recently, the isolation and genetic characterization of mutacin III from the group III S. mutans strain UA787 was reported. Qi et al. (1999) Appl. Environ. Microbiol. 65:3880-3887. The mature mutacin III is twenty-two amino acids in size, and shows striking similarity with another lantibiotic, epidermin, produced by Staphylococcus epidermidis. Allgaier et al. (1986) Eur. J. Biochem. 160:9-22. The mutacin III biosynthesis gene locus consists of eight genes in the order of mutR, —A, —A′, —B, —C, —D, —P, and T. The genomic organization and primary sequence of mutacin III places it in subgroup AI with epidermin and gallidemin as its closest neighbors. Applicants disclosed herein the biochemical and genetic characterization of mutacin I. Comparison of the biosynthesis genes between mutacin I and mutacin III reveal striking similarities as well as important differences.

The cloning and sequencing of the novel mutacin I biosynthetic genes by using information from the conserved sequence derived from several other lantibiotics, and the isolation and purification of mutacin I is disclosed herein and provides a novel group of antibiotics which can be utilized as anti-microbial agents against, for example, presently antibiotic resistant microorganisms.

SUMMARY OF THE INVENTION

According to the present invention, an isolated and purified DNA sequence which encodes a lantibiotic, mutacin I, is disclosed. The nucleic acid sequence is set forth in SEQ ID No: 1 and the amino acid sequence is set forth in SEQ ID No: 2. Also disclosed are pharmaceutical compositions containing mutacin I and methods for their use.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended Figures. These Figures form a part of the specification. It is to be noted, however, that the appended Figures illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. 1. (A) The mutacin III biosynthesis genes. The orientation of the genes and their relative sizes are shown. mutA is the structural gene for prepromutacin I, and mutA' has no known function at present. mutB and -C encode the enzymes for dehydration and thioether bridge formation of premutacin I. mutD encodes a flavoprotein possibly responsible for oxidative decarboxylation of the C-terminal cycteine in premutacin I. mutP and -T code for the protease and ABC transporter, respectively, which are responsible for the processing and transportation of premutacin I. (B) Similarity between MutA (SEQ ID No: 5) and MutA' (SEQ ID No: 6). The middle row shows the identical amino acids and the conserved changes (+). Arrowhead indicates the processing site in MutA (SEQ ID No: 5). The leader peptide and the mature peptide moieties were determined based on MutA (SEQ ID No: 5). (C) Effects of mutA and mutA′ mutations on mutacin I production. Cells from an overnight culture plate were stabbed on TH agar plate and incubated at 37° C. for twenty-four hours. The plate was heated at 80° C. for one hour to kill the producing bacteria, then an overnight culture of the indicator strain NY101 was overlaid on top of the plate. The plate was inspected after an overnight incubation at 37° C.

FIG. 2. Similarity between the mutacin I (SEQ ID No: 5) and mutacin III (SEQ ID No: 18) structural gene. The prepropeptides of mutacin I (SEQ ID No: 5) and mutacin III (SEQ ID No: 18) are compared using the sequence of preepidermin (SEQ ID No: 19) as a reference. The identical amino acids shared by all three lantibiotics are labeled with gray boxes, and the amino acids shared by any of two lantibiotics are labeled with an open box. The conserved sequence FNLD, which is shared by all lantibiotics in subgroup AI (29) is underlined. Brackets indicate the pairs of amino acid residues involved with thioether bridge formation in epidermin (1).

FIG. 3. Purification and EIMS analysis of mutacin I. (A) Elution profile of the first round purification of crude extract of mutacin I by reverse phase HPLC. One-ml fractions were collected along the course of elution and tested for antimicrobial activity (insert). (B) Elution profile of the second round purification using pooled fraction 6 from the first pass as starting material. Fractions 6 and 7 were active. (C) Electrospray ionization mass spectrometry (EIMS) of the purified mutacin I. The mass to charge ratio (m/z) for the doubly-charged molecule (1183) and the triply-charged molecule (788) are labeled. The estimated molecular weight was 2364 Da.

FIG. 4. Biochemical characterization of mutacin I. (A) EIMS analysis of the ethanethiol-derivatized mutacin I. Peaks 1 and 2 are the doubly-charged molecule of 1791 Da and 1774 Da, respectively. The 1774-Da molecule may be a deaminated form of the 1792 Da molecule. Peak 3 may be a deaminated form of peak 4, both of which are singly charged. Peak 5 and peak 6 are triply-charged and doubly-charged molecule of 2719 Da, respectively. Peak 7 is a doubly-charged molecule of 2736 Da, which gives rise to the deaminated form of 2719 Da (peaks 5 and 6). Peak 8 is a singly-charged, deaminated form of peak 9, which has a molecular mass of 1793 Da. The expected molecular mass of mutacin I after insertion of six molecules of ethanethiol is 2736 Da (2364+62×6), which correlated very well with the measured mass of 2736 as shown by peak 7. Addition of the two molecular masses of 1791 (peak 1) and 965 (peak 4) results in a molecular mass of 2756 Da, which would correlate well with the intact modified mutacin I of 2736 Da plus one molecule of H₂O (from breakage of the molecule). (B) Proposed structure of mutacin I based on the data presented in (A) and in FIG. 2. Arrowhead indicates the position where the peptide bound is broken in the ethanethiol-modified mutacin I. The calculated molecular mass for each fragment is labeled. (C) EIMS analysis of mutacin III derivatized with ethanethiol under the same conditions as for mutacin I. The expected molecular mass for fully derived mutacin III is 2636 (see Table 1), and the measured molecular mass is 2638 from the doubly and triply charged peaks (peaks 2 and 3). The 2620-Da molecule as shown by peaks 1 and 4 are probably the deaminated form of the 2638-Da molecule. The 2576-Da molecule as shown in peak 5 resulted from addition of five molecules of ethanethiol (see Table 1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated and purified DNA sequence (SEQ ID No: 1) encoding for a novel lantibiotic, mutacin I, that has been isolated and characterized from Streptococcus mutans CH43.

Further, the present invention provides the isolated and purified DNA sequence for mutacin I designated as mutA (SEQ ID No: 1) and polymorphisms thereof specific for mutacin I.

By “isolated” it is meant separated from other nucleic acids found in bacteria. By “specific” is meant an isolated sequence which encodes the protein mutacin I.

Further, the present invention provides the amino acid sequence of the mutacin I structural protein SEQ ID No: 2, designated MutA and also referred to herein as mutacin I, functional variants thereof. The mutacin I protein has a molecular weight of approximately 2364 Da and is comprised of twenty-four amino acids in its mature form.

Modification to the nucleic acids of the present invention are also contemplated as long as the essential structure and function of the polypeptide encoded by the nucleic acids are maintained. Likewise, fragments used as primers or probes can have substitutions as long as enough complementary bases exist for selective, specific hybridization with high stringency.

Polymorphisms are variants in the gene sequence. They can be sequence shifts found between various bacterial strains and isolates which, while having a different sequence, produce functionally equivalent gene products. Polymorphisms also encompass variations which can be classified as alleles and/or mutations which can produce gene products which may have an altered function. Polymorphisms also encompass variations which can be classified as alleles and/or mutations which either produce no gene product, an inactive gene product, or increased levels of gene product.

The present invention also includes vectors including the mutacin I genes disposed therein. Such vectors are known or can be constructed by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences. Other beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form. Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteriophage vectors. Examples of other vectors include viruses such as bacteriophages, baculoviruses, and retroviruses, DNA viruses, cosmids, plasmids, liposomes, and other recombination vectors. The vectors can also contain elements for use in either prokaryotic or eukaryotic host systems. One of ordinary skill in the art will know which host systems are compatible with a particular vector.

The vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992) and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, Mass. (1988); and Gilboa et al. (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Introduction of nucleic acids by infection offer several advantages over other listed methods. Higher efficiencies can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. The viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.

The above discussion provides a factual basis for the preparation and use of mutacin I (SEQ ID No: 2). The methods used with and the utility of the present invention can be shown by the following non-restrictive examples and Figures.

DNA segments encoding a mutacin gene can be introduced into recombinant host cells and employed for expressing a mutacin I protein or peptide. The introduction of the mutacin I expressing DNA can be accomplished, for example, by the introduction of an organism transformed with the mutacin encoding DNA to act as a probiotic and produce the mutacin I in situ to protect against pathogens or other undesirable organisms. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected mutacin I genes can be employed. Equally, through the application of site-directed mutagenesis techniques, one may re-engineer DNA segments of the present invention to alter the coding sequence, e.g., to introduce improvements to the antibiotic actions of the resultant protein or to test such mutants in order to examine their structure-function relationships at the molecular level. Where desired, one may also prepare fusion peptides, e.g., where the mutacin I coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for immunodetection purposes (e.g., enzyme label coding regions).

Pharmaceutical Compositions and Formulations

Because of the broad spectrum of activity of mutacin I against a variety of microorganisms, mutacin I can be employed to treat multiple drug resistant bacteria such as certain strains of S. aureus which are known to be multiple drug resistant.

Pharmaceutical compositions comprising the disclosed mutacins may be orally administered, for example, with an inert diluent or with an assimilable edible carrier or they may be enclosed in hard or soft shell gelatin capsules or they may be compressed into tablets or may be incorporated directly with the food of the diet.

A therapeutically effective amount is an amount of mutacin I polypeptide (SEQ ID No: 2), the pharmaceutically acceptable salts, esters, amides, and prodrugs thereof, that when administered to a patient or subject, ameliorates a symptom of the condition or disorder.

The compounds of the present invention can be administered to a patient either alone or as part of a pharmaceutical composition.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral prophylaxis, the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes, dentifrices or chewing-type gums. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The active compounds may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally, e.g., formulated for intravenous, intramuscular, or subcutaneous injection. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.

Intravascular devices, such as catheters, have become indispensable tools in the care of seriously ill patients. It is estimated that in the United States alone, 150 million catheters are purchased each year. However, due to the morbidity and mortality resulting from catheter-related infections and the high cost of managing such complications, the benefit derived from these devices may be offset. It has been shown that bloodstream infection due to the use of intravascular catheters (IVC) increased dramatically during the last ten years. From 1975 to 1977, an estimated 3% infection occurred among IVC users, while in 1992 to 1993, this rate increased to 19%. The death rate from such infection is ˜8000 to 16000 per year, exceeding the death rate for AIDS. The cost for treating IVC-related infections is ˜132 to 1600 million per year.

Most infections came from the human skin and the hub of the catheter. Among the infectious bacteria, 40% are coagulase-negative staphylococci, such as S. epidermidis, and 14% are coagulase-positive S. aureus. The remaining are mostly other gram-positive bacteria such as bacilli and enterococci.

Prophylactic methods have been developed to prevent IVC-related infections. The first line of treatment is to sterilize the insertion site with iodine and 70% ethanol. However, compliance with the written protocol is low; only 23% operations follow the protocol. Another preventative measure is the use of catheters impregnated with antibiotics or antiseptic agents such as chlorhexidine and silver sulfadiazine. In clinical trials, mixed results were obtained using such catheters. In addition to the problem of drug resistance by the infecting bacteria, the antibiotics coating the catheters can also be washed away by body fluid, as the attachment of antibiotics to the catheter surface is mainly through ionic interactions.

Because of the urgency to solve the problem of IVC-related infection and the growing market for development of catheters resistant to bacterial attachment on the surface thereof, mutacins are an excellent choice for prevention of IVC-related infections. Mutacin I has the following advantages over conventional antimicrobial agents: 1) it has a wide spectrum of antimicrobial activity against a wide range of gram-positive bacteria including the multidrug-resistant Staphylococci and Enterococci, the major culprits of IVC-related infections; 2) due to its unique mode of action against the sensitive bacteria, resistance to mutacin has not been observed; 3) mutacin is highly thermostable and works in a wide range of pH which makes it suitable for use in a wide range of conditions; 4) its hydrophobic nature can be advantageous for coating the surface of catheters and preventing adhesion of bacteria to the surface; and 5) because it is produced by a normal member of the human oral biota, it is unlikely to elicit immune response from the patient or has any toxicity to the host.

Active mutacin I compound can be coated onto intravascular devices and/or linked to polymers used in the manufacture of these devices to be used to prevent infection caused by intravascular devices. The mutacin I compounds of the present invention can be utilized alone in combination with at least one other entity, such as linked to a polymer, for the prevention or reduction of infection associated with a variety of medical devices such as indwelling tubes or catheters, artificial valves, pacemakers, implantable devices, etc., by incorporating, coating, or otherwise combining the active mutacin I compounds with the materials comprising the patient contact portions of the medical devices. The polymer can be a hydrophobic material or matrix that can be attached to an indwelling device such as a catheter through hydrophobic bonding or can be tethered to the indwelling device through a molecular linker. The incorporation and/or combination of the active mutacin I compounds may be accomplished by coating the medical devices with active mutacin I compounds or by incorporating the active mutacin I compounds into the structure of the medical device. Because the mutacin I compounds of the present invention are very heat stable, they are able to withstand the conditions associated with their incorporation into the medical devices. By combining and/or incorporating the active mutacin I compounds of the present invention into medical devices, both active and passive infection control can be achieved at sites or for uses, which, in many instances, are highly susceptible or vulnerable to infection.

The term “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See, for example, Barge et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66:1-19 which is incorporated herein by reference.)

Examples of pharmaceutically acceptable, non-toxic esters of the compounds of this invention include C₁-C₆ alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C₅-C₇ cycloalkyl esters as well as arylalkyl esters such as, but not limited to benzyl. C₁-C₄ alkyl esters are preferred. Esters of the compounds of the present invention may be prepared according to conventional methods.

Examples of pharmaceutically acceptable, non-toxic amides of the compounds of this invention include amides derived from ammonia, primary C₁-C₆ alkyl amines and secondary C₁-C₆ dialkyl amines wherein the alkyl groups are straight or branched chain. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C₁-C₃ alkyl primary amines, and C₁-C₂ dialkyl secondary amines are preferred. Amides of the compounds of the invention may be prepared according to conventional methods.

The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compounds of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

In addition, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

The compounds of the present invention can be administered to a patient at various dosage. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to those skilled in the art.

EXAMPLES

Materials and Methods

Bacterial strains and media. The group I S. mutans strain CH43 originated from a Chinese school child as part of a natural history study of human caries. Strain CH43 contains a cryptic plasmid similar to other 5.6-kb plasmids within the S. mutans Group I strains. S. sanguis strain NY101 was used as the indicator for mutacin activity assays. CH43 and NY101 were grown on Todd-Hewitt (TH) plate with 1.6% agar (Difco Laboratories, Detroit, Mich.) unless indicated otherwise.

Cloning and sequencing of the mutacin I biosynthetic genes.

Cloning and sequencing of the mutacin I biosynthesis genes were performed exactly as described previously. Qi et al. (1999) Appl. Environ. Microbiol. 65: in press.

Insertional inactivation. The mutA and mut′ genes were inactivated separately by insertion of a kanamycin-resistant gene cassette exactly as described for mutacin III. Qi et al. (1999) Appl. Environ. Microbiol. 65:625-658.

Isolation and purification of mutacin I. For mutacin production, CH43 was grown on TH/agar plate for one day under anaerobic conditions. The cells were then spread on a PHWP membrane with 0.3 μm pore size (Millipore Corp., Bedford, Mass.) on top of a TH plate containing 0.3% agarose. The plate was incubated at 37° C. for two days anaerobically. The membrane was transferred to a new plate for continued incubation every two days, and the old plate was frozen at −70° C. For mutacin isolation, the plates were thawed quickly in a 60° C. water bath. The liquid medium was separated from the agarose debris by centrifugation and the supernatant was passed through a membrane with 0.45 μm pore size. Mutacin I (SEQ ID No: 2) was extracted with an equal volume of chloroform. Novak et al. (1994) J. Bacteriol . 176:4316-4320. The precipitate was dried under a stream of air and washed once with double-distilled H₂O (ddH₂O). The water-insoluble material (crude extract) was dissolved in 6 M urea and tested for antimicrobial activity by a plate assay after a serial dilution with ddH₂O. One arbitrary unit of activity (AU) was defined as the highest dilution that showed a clear zone of inhibition of the indicator strain NY 101.

For purification, the crude extract of mutacin I (SEQ ID No: 2) was applied to a Source 15RPC column and eluted with a fragmented gradient A (0.1% TFA) and B (0.085% TFA in 60% acetonitrile) using a LKB Purifier (Amersham Pharmacia Biotech, Piscataway, N.J.). The active fractions were pooled and dried in a lyophilizer. The pellet was redissolved in 0.25% TFA and subjected to a second round purification using a fragmented gradient of buffer A (0.1% TFA) and B (0.085% TFA in 80% methanol). The single active peak fraction was collected, dried in a lyophilizer, and used for sequence analysis and electrospray ionization mass spectrometry (EIMS).

Chemical modification of mutacin I. Fifty micrograms of purified mutacin I (SEQ ID No: 2) were dried under vacuum and resuspended in 90 μd of a derivatization mixture consisting of 280 μl ethanol, 200 μl water, 65 μl SM sodium hydroxide, and 60 μl ethanethiol as described). Meyer et al. (1994) Anal. Biochem . 223:185-190. The reaction proceeded at 500° C. for one hour under nitrogen, then stopped by the addition of 2 μl acetic acid. The reaction mixture was dried under vacuum and washed three times with 50% ethanol. The pellet was resuspended in 10 μl of 50% acetonitrile with 1% formic acid for EIMS analysis and peptide sequencing by Edman degradation.

Nucleic Acid accession numbers: The sequence for the mutacin I operon has been submitted to Genbank with the accession #AF207710 (AF267498), also designated SEQ ID No: 3.

Results

Cloning and sequencing of the mutacin I biosynthetic genes. As described previously (Qi et al. (1999) Appl Environ. Microbiol. 65:652-658), while isolating mutacin III biosynthesis genes by PCR amplification using a pair of primers designed based on the conserved sequences among LanA and LanB proteins, the mutacin I biosynthesis genes were isolated using the same primers. Sequencing of the isolated PCR fragment demonstrated a striking similarity between the mutacin I and mutacin III genes. By chromosomal walking, the major part of the mutacin I biosynthesis operon was cloned and sequenced as shown in FIG. 1A. It consists of eight genes in the order of mutR, —A, —A′, —B, -C, —D, —P, and —T, which is possibly followed by the immunity gene mutF (SEQ ID No: 12). As in the mutacin III operon, MutR (SEQ ID No: 4) was the positive regulator for the expression of the mutacin I operon (Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658. MutA (SEQ ID No: 5) and MutA′ (SEQ ID No: 6) showed strong similarity to each other as shown in FIG. 1B. Insertional inactivation of mutA and mutA′ demonstrated that mutA was required for mutacin I production, while mutA′ was not as shown in FIG. 1C. This result suggested that, like mutA in the mutacin III operon, the mutA in the mutacin I operon was likely the structural gene encoding prepromutacin I. MutB (SEQ ID No: 7), -C (SEQ ID No: 8) and -D (SEQ ID No: 10) possibly constituted the modification apparatus for prepromutacin I, and MutT (SEQ ID No: 11) and -P (SEQ ID No: 10) are the ABC transporter and protease for transportation and processing of premutacin I, respectively. Other gene encoded mutacin I peptides include MutF (SEQ ID No: 12), MutE (SEQ ID No: 13), MutG (SEQ ID No: 14), OrfX (SEQ ID No: 15), OrfY (SEQ ID No: 16), and OrfZ (SEQ ID No: 17).

Similarity between mutacin I and mutacin III biosynthesis genes. The overall similarity between mutacin I and mutacin III biosynthesis genes was ˜ 94% at the nucleotide level over the 10 kb operon. However, the differences between the two operons were not distributed evenly among the different genes. For example, from mutR to the region immediately upstream of mutA, the similarity was 99%, while in the mutA and mutA′ coding regions, the similarity was only 89% and 91%, respectively. At the amino acid level, the two MutAs (SEQ ID Nos: 5 and 18) shared 84% identical residues as shown in FIG. 2, and the two MutA's shared 93% identical residues. For MutB and MutC the similarity was 93% and 95%, respectively. An even higher similarity (99%) existed in MutP and -T between the two strains.

Purification of mutacin I. To biochemically characterize mutacin I (SEQ ID No: 2), sufficient amount of starting material is required. Applicants' first attempt to isolate mutacin I from liquid culture failed because no mutacin I was produced in any of the liquid cultures that were tested. A stab culture on THIagarose plate as described for mutacin III was then tried. Qi et al. (1999) Appi. Environ. Microbiol . 65:652-658. Mutacin I (SEQ ID No: 2) was produced on such a plate, however the production level was still too low for satisfactory isolation. Based on the observation that mutacin I could be produced on all solid media plates regardless of the media composition, it was reasoned that the production of mutacin I may be regulated by a cell-density mediated control mechanism similar to quorum sensing. (Kleerebezem et al. (1997) Mol. Microbiol. 24:895-904; Surette et al. (1999) Proc. Natl. Acad. Sci. USA 96:1639-1644). Based on this rationale, a membrane transfer technique as described in Materials and Methods was employed, which resulted in a high level of mutacin I production.

Mutacin I (SEQ ID No: 2) was purified by reverse-phase HPLC as shown in FIG. 3. The active fraction (fraction 6) from the first pass (see FIG. 3A) was collected and subjected to a second round purification using a different buffer B and a different gradient (see FIG. 3B). The active fractions (fractions 6 and 7) from the second pass were dried under vacuum and tested for purity by ElMS analysis. As shown in FIG. 3C, mutacin I (SEQ ID No: 2) was purified to near homogeneity as judged by the lack of significant background peaks in the MS chromatogram.

Characterization of mutacin I by ethanethiol derivatization and MS analyses. The molecular weight of mutacin I (SEQ ID NO: 2) was measured by electrospray ionization mass spectrometry. The mass-to-charge ratio for the doubly-charged molecule was 1183, and that for the triply-charged molecule was 788 as shown in FIG. 3C. Thus the measured molecule mass was 2364 Da. This value was in a good agreement with the calculated value of 2516 Da for the unmodified mutacin I minus six molecules of water (108 Da) and one molecule of carboxy residue (45 Da from decarboxylation at the C-terminal cycteine residue).

The primary sequence of mutacin I (SEQ ID No: 2) contained six serine residues and one threonine residue, all of which were potential sites for post-translational dehydration. To confirm that there were indeed six dehydrated residues in the mature mutacin I, an ethanethiol modification of mutacin I under alkaline conditions was performed. In this reaction, one molecule of ethanethiol could insert into the thioether bridge, resulting in a S-ethylcystein and a cystein, or it could insert into the double bound of a dehydrated seine or threonine to form a 5-ethylcystein or a β3-methyl-S-ethylcycteine. Meyer et al. (1994) Anal. Biochem . 223:185-190; Novak et al. (1996) Anal. Biochem . 236:358-360. Ethanethiol derivatization of lantibiotics has been used prior to sequencing of the other lantibiotic gallidermin and pep5 (Meyer et al. (1994) Anal. Biochem . 223:185-190), and for determination of the number of dehydrated amino acid residues in mutacin II (Novak et al. (1996) Anal. Biochem . 236:358-360). The expected molecular mass of mutacin I after each addition of an ethanethiol molecule is listed in Table 1.

TABLE 1 Expected molecular masses of ethanethiol derivatives of mutacins I and III Expected mass (Da) Mutacin 0* 1 2 3 4 5 6 I 2,364 2,426 2,487 2,549 2,611 2,673 2,738 III 2,264 2,318 2,390 2,452 2,514 2,576 2,638 *Number of ethanethiol molecules added.

Quite surprisingly, none of the major peaks generated after ethanethiol modification of mutacin I had the expected molecular mass as shown in FIG. 4A. A very small portion of the molecules showed a pass of 2736 Da (Peak 7), which could account for mutacin I plus six molecules of ethanethiol (2364+62×6); the result of the molecules were all much smaller than expected. With close inspection and calculations, the identity of the small molecules was determined. As shown in FIG. 4B, it appeared that the majority of mutacin I molecules broke into two fragments after the addition of six molecules of ethanethiol. The larger fragment with a mass of 1791 Da was the N-terminal part from F-1 to N-16, and the smaller fragment (965 Da) was the C-terminal part from P-17 to C-24. This finding was of interest because the closely related mutacin III molecule remained intact after the same modification reaction under the same conditions as shown in FIG. 4C.

Peptide sequencing of unmodified and ethanethiol modified mutacin I. Comparison of mutacin I and mutacin III revealed that mutacin I had seven potential dehydration sites (six serines and one threonine), while mutacin III had six (four serines and two threonines). Interestingly, both mutacins had six ethanethiol additions after ethanethiol modification (see FIGS. 4A and 4C), suggesting that all serine or threonine residues in mutacin III were dehydrated. To determine which serine or threonine residue was not dehydrated in mutacin I, the purified mutacin I was subjected to peptide sequencing by Edman degradation. With native mutacin I, Edman degradation was blocked after the first F residue, suggesting that the second serine residue is dehydrated. Dehydrated amino acids were shown to block Edman degradation in other lantibiotics. Gross et al. (1971) J. Am. Chem. Soc. 93:4634-4635; Mota-Meira et al. (1997) FEBS Lett. 410:275-279; Novak et al. (1994) J. Bacteriol. 176:4316-4320.

To get a complete sequence of mutacin I (SEQ ID No: 2), the ethanethiol-derivatized mutacin I had to be used. Ethanethiol-derivatization of lantibiotics was shown to allow Edman degradation to proceed through the dehydrated seine and threonine residues and thioether bridges in other lantibiotics. Meyer et al. (1994) Anal. Biochem . 223:185-190; Mota-Meira et al. (1997) FEBS Lett . 410:275-279. Since the majority of mutacin I molecules was broken into two fragments (see FIG. 4) during ethanethiol modification, the C-terminal fragment had to be eliminated to solve the problem of having two N-termini in the reaction mixture. After several trials, the C-terminal fragment was eliminated by washing the reaction mixture with 30% acetonitnie. The pellet fraction after 30% acetonitrile wash contained mostly the full-length modified mutacin I and the N-terminal fragment. Sequencing of the pellet fraction revealed the following sequence: F₁-SEC₂-SEC₃-L₄-SEC₅-L₆-SEC₇-SEC₈-L ₉-G₁₀-SEC₁₁-T₁₂-G₁₃-V₁₄-K₁₅-N₁₆-P₁₇-SEC₁₈-F₁₉-N₂₀-SEC₂₁-Y₂₂-SEC₂₃. S-ethylcysteine (SEC) was the product of ethanethiol insertion into the double bond of dehydrated serine, or that thioether bridge in lanthionine. The results revealed that all six seine residues in the mutacin I molecule were dehydrated, and that T-12 remained as a nondehydrated residue. In addition, a closer look at the HPLC chromatogram of the sequencing reaction of mutacin I revealed minor peaks in the sequence of P-x-F-N-x-Y. This sequence correlated with the C-terminal fragment of mutacin I: P₁₇-S₁₈-F₁₉-N₂₀-S₂₁-Y₂₂-C₂₃-C₂₄. This result corroborated the previous assignment for the two peptide fragments generated during ethanethiol modification as shown in FIG. 4B.

The mutacin I biosynthesis genes from the group I strain of S. mutans CH43 were cloned and sequenced. DNA and protein sequence analysis revealed that mutacin I and mutacin III are highly homologous to each other, likely arising from a common gene ancestor. Mutacin I was produced by a membrane transfer technique and purified to homogeneity by reverse phase HPLC. The mature mutacin I is twenty-four amino acids in size with a molecular weight of 2364 Da. Ethanethiol modification of mutacin I revealed that it contains six dehydrated amino acids. Sequencing of the native and ethanethiol-derivatized mutacin I by Edman degradation demonstrated that mutacin I is encoded by mutA, and that the six serine residues in the primary sequence of mutacin I are dehydrated, four of which are possibly involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely possesses the same bridging pattern as epidermin.

A closer inspection of the differences between the homologous genes of mutacin I and mutacin III revealed that they are not all distributed evenly. For MutR, —D, —P, and —T, the homology is over 99% between the two mutacins, while for MutA, —A′,—B, and —C, the similarity varies from 87 to 95%. The distribution of the variations within a protein is not even either. For example, in MutA (SEQ ID Nos: 5 and 18), the leader peptide region was identical between the two mutacins. However, the mature peptide region differed by 37.5% (FIG. 2). More interestingly, the sequence of the mature mutacin III is closer to that of epidermin (77% similarity) than to mutacin I (62.5% similarity), while the sequence of the leader peptide of mutacin III and epidermin are dramatically different as seen in FIG. 2. For MutB, —C, —D, —P, and —T proteins, mutacin I and mutacin III are closer to each other than to epidermin.

The biosynthesis of lantibiotics involves several posttranslational modification steps. Chakicherla et al. (1995) J. Biol. Chem. 270:23533-23539; de Vos et al. (1995) Molecular Microbiol. 17:427-437; Sahl et al. (1998) Annu. Rev. Microbiol. 52:41-79. The first step is the translation of the structural gene message into a prepropeptide. The prepropeptide is then modified by dehydration of serine and threonine residues, and formation of thioether bridges between cysteine and the dehydrated amino acid residues. The prepeptide is then translocated across the cell membrane, where the leader peptide is cleaved off and the mature peptide released to the outside medium.

One advantage of lantibiotics over classical antibiotics is its gene-encoded nature, which means that lantibiotics can be altered with ease by manipulating the structural genes through mutagenesis. In reality, however, the number of mutations that can be made is limited because the production of active lantibiotics depends on correct post-translational modification and processing.

Mutacin I and mutacin III are closely related to each other at both the nucleotide and amino acid levels. Comparison of the mature peptide sequence of mutacin I (SEO ID No: 2) and mutacin III suggests that they may also have the same pattern of thioether bridge formation. Despite all the similarities, some important differences exist between the two mutacins. For example, ethanethiol modification of mutacin I broke the molecule into two fragments between N-16 and P-17 as shown in FIG. 4B, while the same reaction did not affect the integrity of mutacin III as shown in FIG. 4C. Comparison of the two mutacins (SEQ ID Nos: 5 and 18) revealed that the major difference is at the linker region (T-12 to P-17), where mutacin I has the sequence T—G—V—K—N—P, and mutacin III has the sequence A—R—T—G as shown in FIG. 2A. These different amino acid residues, according to the statistical figures of Creighton (Creighton, p. 235, in (ed.) Proteins: Structures and molecular principles, W. H. Freeman and Company, New York), have different tendencies in forming different secondary structures in proteins. For example, N-16 and P-17 in mutacin I are more likely to be involved in forming β-turns, while A-12 in mutacin III is more likely to participate in α-helix formation (Stryer, p. 37, in (ed.) Biochemistry, W. H. Freeman and Company, Biochemistry, New York). More importantly, N-16 and P-17 are absent in mutacin III.

In accordance with the possible difference in secondary and tertiary structures, mutacin I and mutacin III have different hydrophobicity and antimicrobial activity. In reverse-phase HPLC analysis, mutacin I is eluted at a higher acetonitrile concentration than mutacin III, suggesting that it is more hydrophobic than mutacin III. In antimicrobial spectrum assays with a limited set of pathogens, mutacin III is more potent than mutacin I against Staphylococcus aureus and Staphylococcus epidermidis, while both mutacins have equal activities against other pathogens such as enterococci, pneumococci, and Group A streptococci.

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 168 <212> TYPE: DNA <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 1 atgtcaaaca cacaattatt agaagtcctt ggtactgaaa cttttgatgt tc #aagaagat     60 ccaacagata ctactattgt ggcaagcaac gacgatccag atactcgttt ct #caagtttg    120 agtttaacag gggtgaaaaa tcctagtttc aatagttact gttgctaa   #               168 <210> SEQ ID NO 2 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 2 Phe Ser Ser Leu Ser Leu Cys Ser Leu Gly Cy #s Thr Gly Val Lys Asn 1               5    #                10   #                15 Pro Ser Phe Asn Ser Tyr Cys Cys             20 <210> SEQ ID NO 3 <211> LENGTH: 15567 <212> TYPE: DNA <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 3 aaatttgttt tttatactaa aagcgggaat gattcaaaac taaaaaagat aa #acgaagaa     60 ttgaaaaagt gatataatag cacagaagag ggcctttata atgaaaggag ac #tattttga    120 aagtaaatca atcaatggaa ttaggtgaac tttatcgaga attaagaatt gc #tagaggtt    180 tgaagataaa agatatagct tgtaaaaatc tgtccaagtc acaactctct ag #atttgaaa    240 atggacaaac catgttggca gctgataaat tgctattagc tatttcggga at #tcatatga    300 gtttttcgga atttggatat gctttgagcc attatgagga gagtgatttt tt #caaaaggg    360 gtaataagtt atcagaatta tatgtccaga aagatatcaa aggattaaaa aa #gttattag    420 aatttaatga caatcatgag gtatttgatg tctacaatcg tttaaataaa tt #ggttattc    480 aagttactat tcatttgcta gatactgatt acataatatc agatgatgat aa #gaattttt    540 taacaactta tctatataat attgaagagt ggactgagta tgaactttat at #ctttggaa    600 atactatgtc tatattgtca tctgatgatt taattttttt gggaaaagct tt #tgtagaac    660 gtgataagtt gtatatatct cttcctagtc ataagaaaaa tgcagagtta ac #ttttttaa    720 atttaatctt aattttgctt gaaagaaaaa aattatatca agcaatctat tt #tgtagaga    780 atttagagaa attattaaat taccaagata tgtttgcaat aacattttta aa #atttttaa    840 aaaaaattat tacttacttt catgataagt cagtagatat gtctgaatta ga #acattata    900 ttaatatagt tgaagaaata aatcctacga ttgcttcaat tcttaaatct aa #tttgaatc    960 agcttttatc aagttttagc cattaaagcc atcttgataa attttatatc tt #tcatattc   1020 attaaatgtg gagataatga aaaagcaacg gttatgctat cgctgctttt tt #tgtgatta   1080 gaagctatgt tatcatggag ttatagtaat gaaacatagt gacagttcat ca #tttcttat   1140 tataaaagtg gtaataagag aagtggtaaa caaagagtta gtaaaataat ac #gtttaacc   1200 ataatatttc ctcctttaat ttattataag attcaaaaag gtaatattcc ta #tatttgca   1260 aatatgggat aaaataattt taaaaaagca gatttgcaat tttaaaaaaa at #agaggcta   1320 atggtggtat tatattattg taaatatatg tttactcagt aatagtgatt ta #ctattaca   1380 acagattttg ttgttatctt agatatttct gctagcatta gttatctgta ga #tgtactac   1440 ttaataagta tataattata attatataat aactattatc agattaccgt ta #aaagtttt   1500 ctgatatgct tctactgaac aatttacgtt cagttacaca catgaaaaag ga #ggatatta   1560 tgtcaaacac acaattatta gaagtccttg gtactgaaac ttttgatgtt ca #agaagatc   1620 tctttgcttt tgatacaaca gatactacta ttgtggcaag caacgacgat cc #agatactc   1680 gtttctcaag tttgagttta tgttcattag gatgtacagg ggtgaaaaat cc #tagtttca   1740 atagttactg ttgctaagtt gtacaaaaga tttagattgt gtcgcatgtc ag #cggcacaa   1800 tcttttgata ttagagatat taaatatgtt aaacacacaa ttattagaag tc #cttggtac   1860 taaaactttt gatgttcaag aagatttatt tgagtttaat ataacagata ct #attgtact   1920 gcaggttagt gatagtccag gtactcatag taaagtgggt agtttcagta tc #tgtcctcc   1980 tcgaaagacc tccgtcagtt tcaatagtta ctgttgttaa ctataaatta ta #cttaaatt   2040 gataggaaac ttggtcatga cattatcata tgttgatatt ggaagagaat ca #aatttata   2100 aagacaatta aatctaaatt tgatgaatat ttagatgaat tattactagg tt #gacagtca   2160 tgttaggaga agagatgaac gattttcaat ttcaagatta ttttatgtac ag #aaaaccat   2220 taggcaactt ttctaatttt cttagtataa ctgatatgat ggatcctatt ga #attattac   2280 ataatgatcc gatatttgct gaaggggtat atttggcttc cccatctctt ag #atcatcta   2340 taaataaatt agagaatcag attgcaagta ctaaggaaaa aaagaatgca aa #agagacta   2400 tttttcaata ctatgcccgt tataacacga gatcaactcc gtttggcttg tt #ttcgtcca   2460 tcggaatagg tggtttttcg aaccacccta ggaaagagaa atcttgttat ga #aaaatctg   2520 ttaatgttga tcttttttgg gcttataaag tagcagataa actagaaagt at #gcctgaaa   2580 ttttaaatac tttaaaagta gttgctaata atgctttgca aaagtcaaat ga #tttttggc   2640 ttttagatac acgaagtcat tttggactta tgaattcacg ttcagatatt cg #tgaggaca   2700 ttacagttaa gtctaatcag cttatagatt atgttattaa ttgcacagaa ga #accaatta   2760 gctatcaaac attaattgat gatattgccg agaaattctc tcaatctagt ga #tgatgtaa   2820 aagaatattt gcaaacatta attaaagagg agtttttaat aactgaattg aa #atttagtt   2880 tgattgatga taatcctttg gattggttta ttaatatttt agaaagagat ca #aaataact   2940 cagaattact tgaaaagttg actgaaataa aggcaatgat tcaagattat ac #tgaccgta   3000 acataggtga aggtaacaat tcgattttag ctctagaaaa taagatgagc ca #aatagtaa   3060 aagccaacgc atacctgcga gttgatcttt atgatcatgc agagctgaag tt #agcgcaac   3120 ataccaagag ttctcttcag aatattttga aagtactaag ttctttttcg tc #agctgtta   3180 atagtcaaaa agaaattaaa aattatcatg agaaatttat tgccaggtat gg #atacgagc   3240 agttagtacc tcttcaatta cttttgaatt ctactagtgg acttggtttt cc #aaaagggt   3300 atagtcaaac agaagtttct aaacaaaata atgaagatag taaaaatcaa aa #aataatag   3360 aatttttaca gagaaaattt gaaaaagctt taagagatgg taaagaaatt at #tttgagtg   3420 atgatgattt aaaagattta aattttgaca cggaacagca aatatcagga ga #attatatt   3480 gtttctacaa ttttaaaagt aaaaagctag aggttagtag tttaggtgtc tc #acagatgc   3540 ttggaaatac ttttggacgt ttccattcta aattgccgaa tacgatagtc ac #aaaaaatg   3600 taaataagac gaaagaaatt tttactgagg cttatccaaa tactattatt ac #tcaattaa   3660 atgaagtgcc atattttggg agaggtggca atattatgat tagtaatagc ct #taaaagtc   3720 accagttgga attgaggaac tatactacta aaaaagagat gagtatcaat ga #tatttatg   3780 tacgtgcaac cagtgaggag ttatattttt attctaagaa atatgagaaa ag #agttattt   3840 ttgtgatgaa taatatgttt aattatataa atggttctaa actcttacgt tt #tttactag   3900 aagtttcaaa ttctgatttt caaaatatta ccccgattac gcttggtagt ct #ggattctt   3960 ataatcatgt gcccgctatc atttataaag atattattat taaaccggaa ac #atggaaca   4020 ttagaaaatc tgaagctaag actttagatt ctctcaaaaa ttggctaact aa #taataatg   4080 ttccgccttt tgtacggatg aaatatactg atcaaattat ttatttagat tt #gagtcgga   4140 ctattgattt aactatgcta tttcagagta tcaaaaaaca tagcttcata ca #attattag   4200 atgttcattc agtatgtaca aacgatacgg agattttaga attagttgtt cc #ttttacaa   4260 gaagtgatgt taacgctcac cagatttatc attatgctca gaatatttat ac #tttggagg   4320 attcaggtag taaagaaaaa tatttttacg ctaaaattta tgtgaataaa ca #acgacaga   4380 cctctttcct acaaaaagag tatcctttat tattaaaata tttgaaactc cc #agaaaact   4440 tacaatggtt ctatattaga tataaagatg atggaaaaga cagcatacgt ct #cagaatca   4500 gatatgtaga agataaacaa ttagttcaac tttattcacg ctttatagag tg #ggcaacaa   4560 aagcacggaa aaatatccaa atttcaggtt atgaaattag tgaatatatc cc #tgaatcag   4620 caagatatgg agggaaaaaa tattcttcaa ttattcattc ttttttctat ta #tgatagta   4680 ttttggattt gcttttacag aagaaagcag aacaaactat tgaagtaaga ac #atctctca   4740 gtattattcg tatgttttta atgatgaaat taagcttaca agaccagcag aa #actcataa   4800 agaatttatt tgatggaaaa cataaactta aatatgaaaa agaatatcat aa #ttcaataa   4860 gtttattact tgacaattta tgtacaaaaa atcagacaga tgaagctgat at #tttctgtg   4920 taatgaatat gaaaaaaatc actgaaaaaa ttagctcagt tcttaaacaa aa #ggacttaa   4980 caacagattg gcagagaatt ctaggaagtt taattcatat gcgatgtaat cg #agtatatg   5040 gaattaacag tgagttagaa agaaaaacaa tgtttattgt tgacaaagtt at #taattcaa   5100 aaagatatac ggatatgttt ttggaggtgg gtaatgagac aaagtaaacg tg #tcgaaaaa   5160 attaaagata ttctaactga gcaaacttat ttattcgatt atcaagaaat at #taaaaaaa   5220 gtcagtcaag caaaacaaac agatttttgg aatttacttt ccttatcttc gg #gaataact   5280 tctttattaa tattttatca agagtatgag aatttagaag gagtaaactt aa #agcagcaa   5340 aagcagtcat taattgggct tataagtcat tatattaatc aaatagcaga ga #aatcctct   5400 ttatttgatg gtttagctgg ggtaggtttt gctattaatt atatctctaa ta #acggtaaa   5460 tattatcaaa aacttcttga acagattgac aacagactcc gtcagaatat tg #aacggaac   5520 cttgtcaact ataagaatga ggaatatgca aatcctatga attatgatgt ag #tttctgga   5580 aatgctggag tagctcgcta cttgatggaa agagaatcct ctgaagattg gc #gaatagtt   5640 gaaatgattt tagaaacatt ttataaagct ttagagcaag gctggcgagt ac #agtcaaaa   5700 tatcaatttc tagagtctga aaagcagtat tatctagaag gaaatataaa tt #tcgggttg   5760 gctcatggaa tattaggacc tgcgacaatt atggctcttt atcaacgaag ag #aaccacaa   5820 aatacaagaa atgctgagaa gcttcaagaa acttatcgac taataaaaag at #acgcccag   5880 gtaagagatg aagggttacg atggccaata cgatatgatt tgtttcgtaa ag #agggttct   5940 tttatattac gaaatggttg gtgttatggc gagaatggca tttataatac ac #tttttctt   6000 atgggaaaag tactctcaaa tcaggagatt tgtgaaactg ctcagaaagt ta #taccatcc   6060 atcataaaag atgattatga gaaaatggaa agtccaacat tttgtcacgg gt #ttgctgga   6120 aaagcaaatt tctttcttct gcaatatcaa agaactaaag aatcaatatt tt #tagttaaa   6180 gcagaagaag aaattgataa aatattaatt gtgtacaatt ctgaaaatat gt #ttggattt   6240 aaagatatag aagataatat tgataatact ggagagagat taacttattg gg #ataatttt   6300 ggtcttctta gtggaactgt tggtgttcta ttagttttga tggaatattg ta #atattgta   6360 aatgccggaa aaattgcaga gtggaataaa atttttcttt tgacttaatt aa #ctgaacgg   6420 agaaataatt atggaagaac aaaatataga gaaaaaaatt ctcttgtgcc ta #acaggttc   6480 tggagcattg ttagggatag ctgaatatat tacgtttttg actgtgcgct tt #aagcatgt   6540 tcgagttatt gtctctgata atgctgcgaa gatgcttcct gttgctgcta tt #acacaatt   6600 gtgtgagaaa gtgtatactg atgaagtttc ctttacagat aagcaaaaga at #cacatagc   6660 tttaactcgc tgggcagaca taacagttgt cttacctgct acagcaaata ta #attggaaa   6720 agttgctaat ggtattgcag ataactttat gacaacaact cttctttctt ct #agcaagcc   6780 agttttaatt tatccttgca tgaataatat tatgtgggaa aatccagtag tt #caaaaaaa   6840 tgttgaagtt ttatctggaa cccaatataa ggtaattgtt ggacaagaat ca #gaatcttt   6900 tgaattagcc agtggaaaga tgaaaaagaa tattgcaatt ccaagtttgg at #gaattgca   6960 acgagttgtt ttagaaaatt tacaagaaga gaggtaagag tatgaagaag aa #aggattac   7020 tagtaataat ctttctaact ttctttttct tttatcctaa agctaaagct gc #tgaatata   7080 caattatatc aaataatagt gaacaaactg ttaatgactt gaataattta gg #agttacag   7140 tcaatagcca tattgcggaa attggatata ttgaagctca aggagatgtt aa #cattgatc   7200 agattaaaaa gctgtcaaat attcaaagta tccagaatat ggctgataca tc #acagaata   7260 tcacgactag agttccttca acatatatta accagacaat acaattgcct ca #gctttttt   7320 cttatcagtg ggatatgcaa aaaattacta ataatggtgt ttcatattca tt #aaataaag   7380 aaaatcgaaa aaatgtaaca gttgctttag ttgattctgg gattgatgta ga #ccataatg   7440 cttttacagg aatgattgat agtcgttcaa aaaattttgt gcctgctgga gg #atatgata   7500 atagtgaaag cagtgaaact ggaaatatta atgatattga tgataaaaaa gg #ccatggaa   7560 cagcagttgc tgggcaaatt gctgcaaatg gtcaaatctt tggtgtgtcc cc #aggaacga   7620 accttcttat ctatagagtt tttggaaaat caaaatcaaa ggagtgctgg at #tttaaaag   7680 caattattga tgcaacaaat aacggtgcta atgttattaa tctaagtttg gg #gcaatata   7740 ttaagattcc taatggtgat atttgggagt ctgccgaagc attaggatat aa #gtttgcca   7800 ttgattatgc cacaagacat aatgtcattg ttgtagcagc cacaggtaat ga #tggattaa   7860 gtgatgacaa cggagaggtt aaaacttatt ataatagtca gcattcagga ca #agatatgt   7920 ctcaaaatga cacggttgaa gattatcctt ctgttttacc taatgctatt gc #agttggct   7980 cttctgataa taataatcaa agatcatctt ttagtaatta ctataatcaa ta #tcaggaca   8040 attttatttt ggctcctggt ggtggaacaa ctttactaga ccaatatggt ca #agaagagt   8100 ggtataatca gaaacttttt atgaaagaac aagtcttatc aacaagtaat aa #tggaaatt   8160 atgattatgc agatggtact tctatttcaa caggaaaagt ttctggagag ct #tgcagaaa   8220 ttattagtaa ctaccatctt caaggagatt cttcaaaagc tagaagtatt ct #actaaatc   8280 aagttaatta tactagtgat ggttataaag aaataagcac ttacaaagct tt #gcgaggtt   8340 actaaatgaa gtggttagaa gttttgcaaa ttagtaaaaa agaaaaaatt ct #ttatctta   8400 ttggttgtat attttcaatt atgacaggct taattactct acgaatcacc ta #cttactta   8460 agaatttagt tgacagcaaa tcgtctttta ataatttgtt cttgtttctt gt #tttgggat   8520 tagttctttt tatcatagat gctggttcac agtatctaat ttcattgatt gg #taatcaag   8580 tagtgtttaa cagtcgaaat aatatttgga aaaaaatttc tgattggaca ga #tagtaaag   8640 atgattcttc tgaaatggca ggccacctta ttaatgatag tgaactgata ga #aaatttta   8700 taatttctac tattcctcaa tcaataaatt cagttattgt tggatcagga tc #cttagtta   8760 tgctatttgt tattaatagt aaaatgtctt tagaagttat agggatttgc tt #gcttttat   8820 tgttcattat gcaacccttt tctagaatat taagcaaaat aagtaaaaga at #ccaggaag   8880 acaaagctga acttattaat attgcctcac agttgagagg acaagtcaaa ac #aataaaaa   8940 gctataatgc tcaagattat gcctttcaaa aatttgatga gcaaaatcgc ca #attatttc   9000 aagatatctt aaatagaata aaaattttta gcatttactc tcctttttta aa #tatcttaa   9060 ttctttttat gattataatt gttgtttggc taggaaatac agaagtacgt tc #aggaaatc   9120 tcactgtagg ttcagcaact atttttgttg tttatatgac acaattaatt aa #tccaatta   9180 tgcaattatc acaattagtt gctcatatgg ggatgcttaa tggcggcgtg ga #acgtcttt   9240 tggagtataa tcaagctatt ccagaaaaaa atggaatcaa gaaaattgat ga #aataatta   9300 atatcgcgtt tgataatgtt tcatttgctt atgataacca agaaaatatt at #tgaaaatg   9360 tgaatttaac ttttcaaaaa ggtacttata tttccattgt tggtgaaagt gg #agttggga   9420 aatcaacctt acttgatctt ttagaacata attatgtacc atcaaaagga cg #aatcttaa   9480 taaacggaat agacttagaa gaattgaata ttaagacttt gcgaaataag at #aagctatg   9540 tatctcaaga accaacaatt ctttctggga caattcgtga actattagac tt #taatcagc   9600 aacagcatac agaaactagt ctttggaatg ttcttgatac tgtagaatta tc #agaactta   9660 ttagaaattt acccgcgaaa ttagattcta aggttgatga atatggtggt aa #cctctctg   9720 gaggtcagat gcaacggatc tcacttgcaa gaggattact gaaagcagga ga #tgttttat   9780 tattagatga atcttttgcc aatattgatg aagagacttg tcttaaaata aa #attaaaaa   9840 ttgctgctta tgctgaatca cacaagcaaa ttgttattga agttattcat aa #tctaaata   9900 gaataactcc cagtagtatc gtttaccgat tggctgataa aaaactagaa at #tttgagga   9960 gcggatttta atagaaaagt cgaagaaatc tgagtaaaag atcagtttct gg #tcgaaaat  10020 taaatattgt gatatataaa taagcttaaa atcaatattc ctaataattt ga #ttttaagc  10080 tttttactat ttgatgagtt tttactcaag atcttttgat tttcctgata aa #gtccttaa  10140 atttgttttt tatactaaaa gcagaaaagg aggatatcat aatggattat at #gctagaga  10200 cgaaaaattt aactaaacag tttggtaagc aaacagcggt taaccaattg aa #tttgaaag  10260 ttgaacgtca ttcaatttat ggtttgctgg ggcctaatgg ttccggcaaa tc #aacaacac  10320 ttaaaatgat tactggaatg ctaagaaaga catctggtca cattcttata ga #cggacacg  10380 attggagccg caaggattta gaaaatatcg gggctctgat tgaatcaccg cc #gctttatg  10440 aaaacctgac tgcgcgtgaa aatttaaagg taagaacctt gatgctgggt tt #acctgata  10500 gtcgcattga tgaggtttta aaaatagtgg atctaaccaa cacgggtaaa aa #aagagcag  10560 ggcaattttc tatgggcatg aagcagcgtc tgggtattgc tatcgcactt tt #gaactcac  10620 ctcaactttt gattctggat gaaccgacta atggacttga tcctattggt at #tcaggagt  10680 tgcgtaatct tattcgttcc ttccctacac aaggaattac agttattatt tc #cagtcata  10740 tcttatctga gattcagatg acagcggatc atattggtat cattgctaat gg #cgtactgg  10800 gttatcagga tagaattcac caagatgaag acttggaaaa actttttact ga #tgtggtta  10860 tgagataccg aggaggtgag tgatatgctg ggcatgtttc aggcagaaag gt #taaaactg  10920 aagcgaagta tggcgaagaa gttactagtt tttgccccca taatagctat tt #tatatggt  10980 tttatagcac ctgtggggta tttagtaaat aatgcttata attggtggta tg #tcatgatt  11040 tttccagggc tgctaacctt atttgctgct ttaataaata cttacgaaga aa #aaaagctg  11100 cattatcgag cagtgtttcc tttgcccatt tctttaagaa aattttggtt tg #aaaaaatt  11160 tttataactg tttattatct taattttagt aatggagtac tttggataat ta #cagtatta  11220 ctgaatactt ttattttacc aaattatgga aaagactata cttatactgt tg #gagaatta  11280 gcactagctt ctttggttat aatagttact acactttggc aaattccatt tt #gtctgtgg  11340 ctgacaaaaa gaatcggttt taccataacg ttgataatta atttaatgag ta #atttcatt  11400 ttgggagttg tttttgcaac tacttcctgc tggtggcttt gtccatatag tt #ggggaata  11460 cgattaatgg tacccatttt aaaaatacta ccgagtggtc taaaggcagg ta #tagcagga  11520 gctccatcat tgccaacaag tttttggagt atcgttatta gtttgtgttt ag #cggttatc  11580 ttatttgtta gtttgacagt tttgagtgca tcttggtttg aaaaacagga ag #tgaaatga  11640 tgattgattt attaaaagca gaaaatgtaa aataccgtca tactttttta cc #atggttac  11700 acctgatttt acctgttact acagctattg ttgttattgt ttatgggcta at #gacgccga  11760 ctcactcttg ggctgatatt actggtggtt acttagaact attgggtata ag #ttttccaa  11820 ttgtcattgc tgttatttgt gggaaatcag ttggactaga agtagaggct gg #tcaatttc  11880 aagttatgtt agcaattaag caaaggaact tgatattttg tatcaagtta tt #gaatttgc  11940 tcattttaga acttttttca actctattag ctataggaat ttatggatta at #ttatcaat  12000 taagtaataa acatttgata ttttatggat atgctgtaat tttactaaca gc #ttcaatgc  12060 tcattcttta tctgattcac ttagttgtag tatttttgtt tggcaatagt gc #taatattg  12120 ggttggggat tgctgaatct ttactatctg ctttgctctt gacaggttta gg #agatggta  12180 tctggcaatt tattccttgt gcttggggta ctcgcctaat gggtacctta at #aaatctgt  12240 ggtattactc tgggcacagc ttatttttta agcaacagct tttaatttgg ct #ggaagtcg  12300 cagttccact aactttaatg gctttaatcc ttagtataat ttggttcgac ag #atggcaag  12360 gacgtagcag tgatgaataa aggaaaaagg agaactttca aacatgacct at #attggtgt  12420 tagtcatctc aaaaaggtgt ataaaactca ggaaggcctc actaacgaag cg #ttaaaaga  12480 tattacgttc tcagttcaag aaggggaatt tattgctatt atgggtgaat ct #ggctcagg  12540 gaagtcaact ctccttaata tcctagcttg tatggattat ccaagtagtg gt #catatcat  12600 cttcaataac tatcaattag agaaagttaa agatgaagag gctgctgttt tt #agaagtcg  12660 gcatattggt tttatttttc aaaatttcaa tcttttaaat atcttcaata at #aaagacaa  12720 tctgttgata ccagttatta tttcgggaag taaggtgaat tcctatgaaa aa #cgattacg  12780 tgatttagct gctgttgttg gtatagaatc tttgctatct aaatatcctt at #gaattatc  12840 tggaggtcaa caacaaaggt tagctattgc cagagcttta attatgaatc ca #gacttgat  12900 attggccgat gagccaacag gacaattgga ctctaagact tctcagcgaa tc #ttgaattt  12960 gttgtctaac atcaacgcta aacgaaagac aattctaatg gtgactcata gt #cctaaagc  13020 tgctagttat gcaaaccgag ttctttttat caaggatggt gttattttca at #caacttgt  13080 tcgtgggtgt aaatccaggg aaggcttttt agatcaaatt attatggctc ag #gccagtct  13140 gtaggaggtt gtcctgatta tgtttttacc caaaatttcc tttcataatc tt #attgtaaa  13200 taaatcatta accttacctt attttgctat tatgaccatt tttagtggtt tt #aactatgt  13260 tttgattaat tttttaacca accctagttt ttataacatt ccaacagcta gg #atactgat  13320 tgatattctt atttttggtt ttatcttaat ttcattactg atgttgcttt at #ggtcgcta  13380 tgccaatcgt tttataagtg atgagcgtaa tagtaatatg ggaatttttc tc #atgttggg  13440 aatggggaaa aagcaattat taaaaataat ctatttggaa aagttatatc tt #tttacagg  13500 aacgtttttt ggaggtttaa tctttggttt cgtatacagt aagatatttt tt #ctttttat  13560 cagaaatcta attgttattg gagatgtcag agaacaatat agcttaacgg ct #attagttg  13620 gctacttatt cttacttttt ttatttattt tattatttat ctatcagagt ac #cgattatt  13680 aaaacgtcaa agtatcacgg ttatttttaa tagcaaagct aagcgtgata at #cctagaaa  13740 aactagtgtt tttgttggac tttttggact ttttgccctg ttaatgggat at #cattttgc  13800 tttaacaagt cccaatgtca caaccagttt cagccgtttc atttatgctg cc #tgcttagt  13860 tactctaggt attttttgca cgttttcgtc aggtgtgatt atgttactga ct #gtcataaa  13920 gaagagaaga gctatctact ataatcaacg gcgctttgtt gtgattgcta gt #ttatttca  13980 ccgtatccgc agtaatgctc tgtctttggc gactatctgt atttttagca cc #gctacctt  14040 agttagttta tctgtcttag ctagtctcta tcttgcaaag gacaatatgg tt #cgtctttc  14100 aagtcctaga gatgttacgg tgctatctac aactgatatt gaaccgaatt ta #atggacat  14160 cgctacaaaa aatcatgtta ctctaactaa tcgccagaat ttaaaggttt ct #caatctgt  14220 ttatggtaat atcaaaggaa gtcatttgtc agttgatcct aatggcggta tg #gctaatga  14280 ttatcaaata acagttattt cattggattc ttttaatgct tctaataata cc #cattatcg  14340 tttaaaaaat catgaaattc tcacctatgt ttcaaatgga gcagctgctc cc #tctagcta  14400 tacaactaat ggtgttaaac taaccaatgt taaacaaatt aaaaggataa ac #tttatttt  14460 ttctccgcta cgctctatgc agcctaattt ctttataatt actgacaatc ga #gaaataat  14520 tcagactatt ttgaaagagg agctaacatg gggaacgatg gcaggctacc at #gttaaagg  14580 aaaaaaaatg aatcagaaag atttttatga tgagcttgag actactaatt tc #aggcaatt  14640 tagtgctaat gtagtttcaa taagacaggt caaatcaatg tttaatgctt ta #tttggcgg  14700 tttactcttt gttggtatta tttttggaac tatttttgca attttgacag ct #ataactat  14760 ttattatcaa cagctttctg aaggaattcg agaccgagat gattataagg cc #atgataaa  14820 attaggtatg acaaataaaa ctattcaaga cagtattaag gttcaaataa ac #tttgtttt  14880 catcttgccc attgcttttg ccctattaaa tctcatcttt gcacttccta tt #ttatataa  14940 aataatgaca acttttggat ttaatgatgc aggactattt ctaagagctg tt #ggaacttg  15000 tctgattgtt taccttttct tttattggtt tatttgtcat tgcacatcca aa #ctatatta  15060 tcgtttaata tctaaaaaat agaggagttt atattatgcg tattgtaagt tc #attggtat  15120 cgcttttatt gactatcttt tggatttttg ctatagcttt tatcccaatt gg #agaccaga  15180 atagttttaa taaaccagaa atgtggttct ttgttttttt cgctattatt at #ttatagta  15240 ttgttataat aagcgattat tatctaaaga gctttaatct tttgaaagtt ta #tcaaattt  15300 tagttttgtt tattagcata ctgtgtgctc tttgtggttt atcactaact gc #tttaggat  15360 tgaaagtatt cactttagct attggaattg ttagtcttgt taatacaatt at #ttatttct  15420 ttttcgctaa taaaaaagat aatgttgaat aaaatatgtt atcctagtga ag #gaggtttc  15480 ctagaatgac ccgtattttg gtaattgatg atgatgcaga tattttggct ct #gataaaaa  15540 ataccttgca actgcaaaac tatctgg           #                   #          15567 <210> SEQ ID NO 4 <211> LENGTH: 289 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 4 Leu Lys Val Asn Gln Ser Met Glu Leu Gly Gl #u Leu Tyr Arg Glu Leu 1               5    #                10   #                15 Arg Ile Ala Arg Gly Leu Lys Ile Lys Asp Il #e Ala Cys Lys Asn Leu             20       #            25       #            30 Ser Lys Ser Gln Leu Ser Arg Phe Glu Asn Gl #y Gln Thr Met Leu Ala         35           #        40           #        45 Ala Asp Lys Leu Leu Leu Ala Ile Ser Gly Il #e His Met Ser Phe Ser     50               #    55               #    60 Glu Phe Gly Tyr Ala Leu Ser His Tyr Glu Gl #u Ser Asp Phe Phe Lys 65                   #70                   #75                   #80 Arg Gly Asn Lys Leu Ser Glu Leu Tyr Val Gl #n Lys Asp Ile Lys Gly                 85   #                90   #                95 Leu Lys Lys Leu Leu Glu Phe Asn Asp Asn Hi #s Glu Val Phe Asp Val             100       #           105       #           110 Tyr Asn Arg Leu Asn Lys Leu Val Ile Gln Va #l Thr Ile His Leu Leu         115           #       120           #       125 Asp Thr Asp Tyr Ile Ile Ser Asp Asp Asp Ly #s Asn Phe Leu Thr Thr     130               #   135               #   140 Tyr Leu Tyr Asn Ile Glu Glu Trp Thr Glu Ty #r Glu Leu Tyr Ile Phe 145                 1 #50                 1 #55                 1 #60 Gly Asn Thr Met Ser Ile Leu Ser Ser Asp As #p Leu Ile Phe Leu Gly                 165   #               170   #               175 Lys Ala Phe Val Glu Arg Asp Lys Leu Tyr Il #e Ser Leu Pro Ser His             180       #           185       #           190 Lys Lys Asn Ala Glu Leu Thr Phe Leu Asn Le #u Ile Leu Ile Leu Leu         195           #       200           #       205 Glu Arg Lys Lys Leu Tyr Gln Ala Ile Tyr Ph #e Val Glu Asn Leu Glu     210               #   215               #   220 Lys Leu Leu Asn Tyr Gln Asp Met Phe Ala Il #e Thr Phe Leu Lys Phe 225                 2 #30                 2 #35                 2 #40 Leu Lys Lys Ile Ile Thr Tyr Phe His Asp Ly #s Ser Val Asp Met Ser                 245   #               250   #               255 Glu Leu Glu His Tyr Ile Asn Ile Val Glu Gl #u Ile Asn Pro Thr Ile             260       #           265       #           270 Ala Ser Ile Leu Lys Ser Asn Leu Asn Gln Le #u Leu Ser Ser Phe Ser         275           #       280           #       285 His <210> SEQ ID NO 5 <211> LENGTH: 65 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 5 Met Ser Asn Thr Gln Leu Leu Glu Val Leu Gl #y Thr Glu Thr Phe Asp 1               5    #                10   #                15 Val Gln Glu Asp Leu Phe Ala Phe Asp Thr Th #r Asp Thr Thr Ile Val             20       #            25       #            30 Ala Ser Asn Asp Asp Pro Asp Thr Arg Phe Se #r Ser Leu Ser Leu Cys         35           #        40           #        45 Ser Leu Gly Cys Thr Gly Val Lys Asn Pro Se #r Phe Asn Ser Tyr Cys     50               #    55               #    60 Cys 65 <210> SEQ ID NO 6 <211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 6 Met Leu Asn Thr Gln Leu Leu Glu Val Leu Gl #y Thr Lys Thr Phe Asp 1               5    #                10   #                15 Val Gln Glu Asp Leu Phe Glu Phe Asn Ile Th #r Asp Thr Ile Val Leu             20       #            25       #            30 Gln Val Ser Asp Ser Pro Gly Thr His Ser Ly #s Val Gly Ser Phe Ser         35           #        40           #        45 Ile Cys Pro Pro Arg Lys Thr Ser Val Ser Ph #e Asn Ser Tyr Cys Cys     50               #    55               #    60 <210> SEQ ID NO 7 <211> LENGTH: 990 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 7 Met Asn Asp Phe Gln Phe Gln Asp Tyr Phe Me #t Tyr Arg Lys Pro Leu 1               5    #                10   #                15 Gly Asn Phe Ser Asn Phe Leu Ser Ile Thr As #p Met Met Asp Pro Ile             20       #            25       #            30 Glu Leu Leu His Asn Asp Pro Ile Phe Ala Gl #u Gly Val Tyr Leu Ala         35           #        40           #        45 Ser Pro Ser Leu Arg Ser Ser Ile Asn Lys Le #u Glu Asn Gln Ile Ala     50               #    55               #    60 Ser Thr Lys Glu Lys Lys Asn Ala Lys Glu Th #r Ile Phe Gln Tyr Tyr 65                   #70                   #75                   #80 Ala Arg Tyr Asn Thr Arg Ser Thr Pro Phe Gl #y Leu Phe Ser Ser Ile                 85   #                90   #                95 Gly Ile Gly Gly Phe Ser Asn His Pro Arg Ly #s Glu Lys Ser Cys Tyr             100       #           105       #           110 Glu Lys Ser Val Asn Val Asp Leu Phe Trp Al #a Tyr Lys Val Ala Asp         115           #       120           #       125 Lys Leu Glu Ser Met Pro Glu Ile Leu Asn Th #r Leu Lys Val Val Ala     130               #   135               #   140 Asn Asn Ala Leu Gln Lys Ser Asn Asp Phe Tr #p Leu Leu Asp Thr Arg 145                 1 #50                 1 #55                 1 #60 Ser His Phe Gly Leu Met Asn Ser Arg Ser As #p Ile Arg Glu Asp Ile                 165   #               170   #               175 Thr Val Lys Ser Asn Gln Leu Ile Asp Tyr Va #l Ile Asn Cys Thr Glu             180       #           185       #           190 Glu Pro Ile Ser Tyr Gln Thr Leu Ile Asp As #p Ile Ala Glu Lys Phe         195           #       200           #       205 Ser Gln Ser Ser Asp Asp Val Lys Glu Tyr Le #u Gln Thr Leu Ile Lys     210               #   215               #   220 Glu Glu Phe Leu Ile Thr Glu Leu Lys Phe Se #r Leu Ile Asp Asp Asn 225                 2 #30                 2 #35                 2 #40 Pro Leu Asp Trp Phe Ile Asn Ile Leu Glu Ar #g Asp Gln Asn Asn Ser                 245   #               250   #               255 Glu Leu Leu Glu Lys Leu Thr Glu Ile Lys Al #a Met Ile Gln Asp Tyr             260       #           265       #           270 Thr Asp Arg Asn Ile Gly Glu Gly Asn Asn Se #r Ile Leu Ala Leu Glu         275           #       280           #       285 Asn Lys Met Ser Gln Ile Val Lys Ala Asn Al #a Tyr Leu Arg Val Asp     290               #   295               #   300 Leu Tyr Asp His Ala Glu Leu Lys Leu Ala Gl #n His Thr Lys Ser Ser 305                 3 #10                 3 #15                 3 #20 Leu Gln Asn Ile Leu Lys Val Leu Ser Ser Ph #e Ser Ser Ala Val Asn                 325   #               330   #               335 Ser Gln Lys Glu Ile Lys Asn Tyr His Glu Ly #s Phe Ile Ala Arg Tyr             340       #           345       #           350 Gly Tyr Glu Gln Leu Val Pro Leu Gln Leu Le #u Leu Asn Ser Thr Ser         355           #       360           #       365 Gly Leu Gly Phe Pro Lys Gly Tyr Ser Gln Th #r Glu Val Ser Lys Gln     370               #   375               #   380 Asn Asn Glu Asp Ser Lys Asn Gln Lys Ile Il #e Glu Phe Leu Gln Arg 385                 3 #90                 3 #95                 4 #00 Lys Phe Glu Lys Ala Leu Arg Asp Gly Lys Gl #u Ile Ile Leu Ser Asp                 405   #               410   #               415 Asp Asp Leu Lys Asp Leu Asn Phe Asp Thr Gl #u Gln Gln Ile Ser Gly             420       #           425       #           430 Glu Leu Tyr Cys Phe Tyr Asn Phe Lys Ser Ly #s Lys Leu Glu Val Ser         435           #       440           #       445 Ser Leu Gly Val Ser Gln Met Leu Gly Asn Th #r Phe Gly Arg Phe His     450               #   455               #   460 Ser Lys Leu Pro Asn Thr Ile Val Thr Lys As #n Val Asn Lys Thr Lys 465                 4 #70                 4 #75                 4 #80 Glu Ile Phe Thr Glu Ala Tyr Pro Asn Thr Il #e Ile Thr Gln Leu Asn                 485   #               490   #               495 Glu Val Pro Tyr Phe Gly Arg Gly Gly Asn Il #e Met Ile Ser Asn Ser             500       #           505       #           510 Leu Lys Ser His Gln Leu Glu Leu Arg Asn Ty #r Thr Thr Lys Lys Glu         515           #       520           #       525 Met Ser Ile Asn Asp Ile Tyr Val Arg Ala Th #r Ser Glu Glu Leu Tyr     530               #   535               #   540 Phe Tyr Ser Lys Lys Tyr Glu Lys Arg Val Il #e Phe Val Met Asn Asn 545                 5 #50                 5 #55                 5 #60 Met Phe Asn Tyr Ile Asn Gly Ser Lys Leu Le #u Arg Phe Leu Leu Glu                 565   #               570   #               575 Val Ser Asn Ser Asp Phe Gln Asn Ile Thr Pr #o Ile Thr Leu Gly Ser             580       #           585       #           590 Leu Asp Ser Tyr Asn His Val Pro Ala Ile Il #e Tyr Lys Asp Ile Ile         595           #       600           #       605 Ile Lys Pro Glu Thr Trp Asn Ile Arg Lys Se #r Glu Ala Lys Thr Leu     610               #   615               #   620 Asp Ser Leu Lys Asn Trp Leu Thr Asn Asn As #n Val Pro Pro Phe Val 625                 6 #30                 6 #35                 6 #40 Arg Met Lys Tyr Thr Asp Gln Ile Ile Tyr Le #u Asp Leu Ser Arg Thr                 645   #               650   #               655 Ile Asp Leu Thr Met Leu Phe Gln Ser Ile Ly #s Lys His Ser Phe Ile             660       #           665       #           670 Gln Leu Leu Asp Val His Ser Val Cys Thr As #n Asp Thr Glu Ile Leu         675           #       680           #       685 Glu Leu Val Val Pro Phe Thr Arg Ser Asp Va #l Asn Ala His Gln Ile     690               #   695               #   700 Tyr His Tyr Ala Gln Asn Ile Tyr Thr Leu Gl #u Asp Ser Gly Ser Lys 705                 7 #10                 7 #15                 7 #20 Glu Lys Tyr Phe Tyr Ala Lys Ile Tyr Val As #n Lys Gln Arg Gln Thr                 725   #               730   #               735 Ser Phe Leu Gln Lys Glu Tyr Pro Leu Leu Le #u Lys Tyr Leu Lys Leu             740       #           745       #           750 Pro Glu Asn Leu Gln Trp Phe Tyr Ile Arg Ty #r Lys Asp Asp Gly Lys         755           #       760           #       765 Asp Ser Ile Arg Leu Arg Ile Arg Tyr Val Gl #u Asp Lys Gln Leu Val     770               #   775               #   780 Gln Leu Tyr Ser Arg Phe Ile Glu Trp Ala Th #r Lys Ala Arg Lys Asn 785                 7 #90                 7 #95                 8 #00 Ile Gln Ile Ser Gly Tyr Glu Ile Ser Glu Ty #r Ile Pro Glu Ser Ala                 805   #               810   #               815 Arg Tyr Gly Gly Lys Lys Tyr Ser Ser Ile Il #e His Ser Phe Phe Tyr             820       #           825       #           830 Tyr Asp Ser Ile Leu Asp Leu Leu Leu Gln Ly #s Lys Ala Glu Gln Thr         835           #       840           #       845 Ile Glu Val Arg Thr Ser Leu Ser Ile Ile Ar #g Met Phe Leu Met Met     850               #   855               #   860 Lys Leu Ser Leu Gln Asp Gln Gln Lys Leu Il #e Lys Asn Leu Phe Asp 865                 8 #70                 8 #75                 8 #80 Gly Lys His Lys Leu Lys Tyr Glu Lys Glu Ty #r His Asn Ser Ile Ser                 885   #               890   #               895 Leu Leu Leu Asp Asn Leu Cys Thr Lys Asn Gl #n Thr Asp Glu Ala Asp             900       #           905       #           910 Ile Phe Cys Val Met Asn Met Lys Lys Ile Th #r Glu Lys Ile Ser Ser         915           #       920           #       925 Val Leu Lys Gln Lys Asp Leu Thr Thr Asp Tr #p Gln Arg Ile Leu Gly     930               #   935               #   940 Ser Leu Ile His Met Arg Cys Asn Arg Val Ty #r Gly Ile Asn Ser Glu 945                 9 #50                 9 #55                 9 #60 Leu Glu Arg Lys Thr Met Phe Ile Val Asp Ly #s Val Ile Asn Ser Lys                 965   #               970   #               975 Arg Tyr Thr Asp Met Phe Leu Glu Val Gly As #n Glu Thr Lys             980       #           985       #           990 <210> SEQ ID NO 8 <211> LENGTH: 424 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 8 Met Arg Gln Ser Lys Arg Val Glu Lys Ile Ly #s Asp Ile Leu Thr Glu 1               5    #                10   #                15 Gln Thr Tyr Leu Phe Asp Tyr Gln Glu Ile Le #u Lys Lys Val Ser Gln             20       #            25       #            30 Ala Lys Gln Thr Asp Phe Trp Asn Leu Leu Se #r Leu Ser Ser Gly Ile         35           #        40           #        45 Thr Ser Leu Leu Ile Phe Tyr Gln Glu Tyr Gl #u Asn Leu Glu Gly Val     50               #    55               #    60 Asn Leu Lys Gln Gln Lys Gln Ser Leu Ile Gl #y Leu Ile Ser His Tyr 65                   #70                   #75                   #80 Ile Asn Gln Ile Ala Glu Lys Ser Ser Leu Ph #e Asp Gly Leu Ala Gly                 85   #                90   #                95 Val Gly Phe Ala Ile Asn Tyr Ile Ser Asn As #n Gly Lys Tyr Tyr Gln             100       #           105       #           110 Lys Leu Leu Glu Gln Ile Asp Asn Arg Leu Ar #g Gln Asn Ile Glu Arg         115           #       120           #       125 Asn Leu Val Asn Tyr Lys Asn Glu Glu Tyr Al #a Asn Pro Met Asn Tyr     130               #   135               #   140 Asp Val Val Ser Gly Asn Ala Gly Val Ala Ar #g Tyr Leu Met Glu Arg 145                 1 #50                 1 #55                 1 #60 Glu Ser Ser Glu Asp Trp Arg Ile Val Glu Me #t Ile Leu Glu Thr Phe                 165   #               170   #               175 Tyr Lys Ala Leu Glu Gln Gly Trp Arg Val Gl #n Ser Lys Tyr Gln Phe             180       #           185       #           190 Leu Glu Ser Glu Lys Gln Tyr Tyr Leu Glu Gl #y Asn Ile Asn Phe Gly         195           #       200           #       205 Leu Ala His Gly Ile Leu Gly Pro Ala Thr Il #e Met Ala Leu Tyr Gln     210               #   215               #   220 Arg Arg Glu Pro Gln Asn Thr Arg Asn Ala Gl #u Lys Leu Gln Glu Thr 225                 2 #30                 2 #35                 2 #40 Tyr Arg Leu Ile Lys Arg Tyr Ala Gln Val Ar #g Asp Glu Gly Leu Arg                 245   #               250   #               255 Trp Pro Ile Arg Tyr Asp Leu Phe Arg Lys Gl #u Gly Ser Phe Ile Leu             260       #           265       #           270 Arg Asn Gly Trp Cys Tyr Gly Glu Asn Gly Il #e Tyr Asn Thr Leu Phe         275           #       280           #       285 Leu Met Gly Lys Val Leu Ser Asn Gln Glu Il #e Cys Glu Thr Ala Gln     290               #   295               #   300 Lys Val Ile Pro Ser Ile Ile Lys Asp Asp Ty #r Glu Lys Met Glu Ser 305                 3 #10                 3 #15                 3 #20 Pro Thr Phe Cys His Gly Phe Ala Gly Lys Al #a Asn Phe Phe Leu Leu                 325   #               330   #               335 Gln Tyr Gln Arg Thr Lys Glu Ser Ile Phe Le #u Val Lys Ala Glu Glu             340       #           345       #           350 Glu Ile Asp Lys Ile Leu Ile Val Tyr Asn Se #r Glu Asn Met Phe Gly         355           #       360           #       365 Phe Lys Asp Ile Glu Asp Asn Ile Asp Asn Th #r Gly Glu Arg Leu Thr     370               #   375               #   380 Tyr Trp Asp Asn Phe Gly Leu Leu Ser Gly Th #r Val Gly Val Leu Leu 385                 3 #90                 3 #95                 4 #00 Val Leu Met Glu Tyr Cys Asn Ile Val Asn Al #a Gly Lys Ile Ala Glu                 405   #               410   #               415 Trp Asn Lys Ile Phe Leu Leu Thr             420 <210> SEQ ID NO 9 <211> LENGTH: 188 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 9 Met Glu Glu Gln Asn Ile Glu Lys Lys Ile Le #u Leu Cys Leu Thr Gly 1               5    #                10   #                15 Ser Gly Ala Leu Leu Gly Ile Ala Glu Tyr Il #e Thr Phe Leu Thr Val             20       #            25       #            30 Arg Phe Lys His Val Arg Val Ile Val Ser As #p Asn Ala Ala Lys Met         35           #        40           #        45 Leu Pro Val Ala Ala Ile Thr Gln Leu Cys Gl #u Lys Val Tyr Thr Asp     50               #    55               #    60 Glu Val Ser Phe Thr Asp Lys Gln Lys Asn Hi #s Ile Ala Leu Thr Arg 65                   #70                   #75                   #80 Trp Ala Asp Ile Thr Val Val Leu Pro Ala Th #r Ala Asn Ile Ile Gly                 85   #                90   #                95 Lys Val Ala Asn Gly Ile Ala Asp Asn Phe Me #t Thr Thr Thr Leu Leu             100       #           105       #           110 Ser Ser Ser Lys Pro Val Leu Ile Tyr Pro Cy #s Met Asn Asn Ile Met         115           #       120           #       125 Trp Glu Asn Pro Val Val Gln Lys Asn Val Gl #u Val Leu Ser Gly Thr     130               #   135               #   140 Gln Tyr Lys Val Ile Val Gly Gln Glu Ser Gl #u Ser Phe Glu Leu Ala 145                 1 #50                 1 #55                 1 #60 Ser Gly Lys Met Lys Lys Asn Ile Ala Ile Pr #o Ser Leu Asp Glu Leu                 165   #               170   #               175 Gln Arg Val Val Leu Glu Asn Leu Gln Glu Gl #u Arg             180       #           185 <210> SEQ ID NO 10 <211> LENGTH: 447 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 10 Met Lys Lys Lys Gly Leu Leu Val Ile Ile Ph #e Leu Thr Phe Phe Phe 1               5    #                10   #                15 Phe Tyr Pro Lys Ala Lys Ala Ala Glu Tyr Th #r Ile Ile Ser Asn Asn             20       #            25       #            30 Ser Glu Gln Thr Val Asn Asp Leu Asn Asn Le #u Gly Val Thr Val Asn         35           #        40           #        45 Ser His Ile Ala Glu Ile Gly Tyr Ile Glu Al #a Gln Gly Asp Val Asn     50               #    55               #    60 Ile Asp Gln Ile Lys Lys Leu Ser Asn Ile Gl #n Ser Ile Gln Asn Met 65                   #70                   #75                   #80 Ala Asp Thr Ser Gln Asn Ile Thr Thr Arg Va #l Pro Ser Thr Tyr Ile                 85   #                90   #                95 Asn Gln Thr Ile Gln Leu Pro Gln Leu Phe Se #r Tyr Gln Trp Asp Met             100       #           105       #           110 Gln Lys Ile Thr Asn Asn Gly Val Ser Tyr Se #r Leu Asn Lys Glu Asn         115           #       120           #       125 Arg Lys Asn Val Thr Val Ala Leu Val Asp Se #r Gly Ile Asp Val Asp     130               #   135               #   140 His Asn Ala Phe Thr Gly Met Ile Asp Ser Ar #g Ser Lys Asn Phe Val 145                 1 #50                 1 #55                 1 #60 Pro Ala Gly Gly Tyr Asp Asn Ser Glu Ser Se #r Glu Thr Gly Asn Ile                 165   #               170   #               175 Asn Asp Ile Asp Asp Lys Lys Gly His Gly Th #r Ala Val Ala Gly Gln             180       #           185       #           190 Ile Ala Ala Asn Gly Gln Ile Phe Gly Val Se #r Pro Gly Thr Asn Leu         195           #       200           #       205 Leu Ile Tyr Arg Val Phe Gly Lys Ser Lys Se #r Lys Glu Cys Trp Ile     210               #   215               #   220 Leu Lys Ala Ile Ile Asp Ala Thr Asn Asn Gl #y Ala Asn Val Ile Asn 225                 2 #30                 2 #35                 2 #40 Leu Ser Leu Gly Gln Tyr Ile Lys Ile Pro As #n Gly Asp Ile Trp Glu                 245   #               250   #               255 Ser Ala Glu Ala Leu Gly Tyr Lys Phe Ala Il #e Asp Tyr Ala Thr Arg             260       #           265       #           270 His Asn Val Ile Val Val Ala Ala Thr Gly As #n Asp Gly Leu Ser Asp         275           #       280           #       285 Asp Asn Gly Glu Val Lys Thr Tyr Tyr Asn Se #r Gln His Ser Gly Gln     290               #   295               #   300 Asp Met Ser Gln Asn Asp Thr Val Glu Asp Ty #r Pro Ser Val Leu Pro 305                 3 #10                 3 #15                 3 #20 Asn Ala Ile Ala Val Gly Ser Ser Asp Asn As #n Asn Gln Arg Ser Ser                 325   #               330   #               335 Phe Ser Asn Tyr Tyr Asn Gln Tyr Gln Asp As #n Phe Ile Leu Ala Pro             340       #           345       #           350 Gly Gly Gly Thr Thr Leu Leu Asp Gln Tyr Gl #y Gln Glu Glu Trp Tyr         355           #       360           #       365 Asn Gln Lys Leu Phe Met Lys Glu Gln Val Le #u Ser Thr Ser Asn Asn     370               #   375               #   380 Gly Asn Tyr Asp Tyr Ala Asp Gly Thr Ser Il #e Ser Thr Gly Lys Val 385                 3 #90                 3 #95                 4 #00 Ser Gly Glu Leu Ala Glu Ile Ile Ser Asn Ty #r His Leu Gln Gly Asp                 405   #               410   #               415 Ser Ser Lys Ala Arg Ser Ile Leu Leu Asn Gl #n Val Asn Tyr Thr Ser             420       #           425       #           430 Asp Gly Tyr Lys Glu Ile Ser Thr Tyr Lys Al #a Leu Arg Gly Tyr         435           #       440           #       445 <210> SEQ ID NO 11 <211> LENGTH: 541 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 11 Met Lys Trp Leu Glu Val Leu Gln Ile Ser Ly #s Lys Glu Lys Ile Leu 1               5    #                10   #                15 Tyr Leu Ile Gly Cys Ile Phe Ser Ile Met Th #r Gly Leu Ile Thr Leu             20       #            25       #            30 Arg Ile Thr Tyr Leu Leu Lys Asn Leu Val As #p Ser Lys Ser Ser Phe         35           #        40           #        45 Asn Asn Leu Phe Leu Phe Leu Val Leu Gly Le #u Val Leu Phe Ile Ile     50               #    55               #    60 Asp Ala Gly Ser Gln Tyr Leu Ile Ser Leu Il #e Gly Asn Gln Val Val 65                   #70                   #75                   #80 Phe Asn Ser Arg Asn Asn Ile Trp Lys Lys Il #e Ser Asp Trp Thr Asp                 85   #                90   #                95 Ser Lys Asp Asp Ser Ser Glu Met Ala Gly Hi #s Leu Ile Asn Asp Ser             100       #           105       #           110 Glu Leu Ile Glu Asn Phe Ile Ile Ser Thr Il #e Pro Gln Ser Ile Asn         115           #       120           #       125 Ser Val Ile Val Gly Ser Gly Ser Leu Val Me #t Leu Phe Val Ile Asn     130               #   135               #   140 Ser Lys Met Ser Leu Glu Val Ile Gly Ile Cy #s Leu Leu Leu Leu Phe 145                 1 #50                 1 #55                 1 #60 Ile Met Gln Pro Phe Ser Arg Ile Leu Ser Ly #s Ile Ser Lys Arg Ile                 165   #               170   #               175 Gln Glu Asp Lys Ala Glu Leu Ile Asn Ile Al #a Ser Gln Leu Arg Gly             180       #           185       #           190 Gln Val Lys Thr Ile Lys Ser Tyr Asn Ala Gl #n Asp Tyr Ala Phe Gln         195           #       200           #       205 Lys Phe Asp Glu Gln Asn Arg Gln Leu Phe Gl #n Asp Ile Leu Asn Arg     210               #   215               #   220 Ile Lys Ile Phe Ser Ile Tyr Ser Pro Phe Le #u Asn Ile Leu Ile Leu 225                 2 #30                 2 #35                 2 #40 Phe Met Ile Ile Ile Val Val Trp Leu Gly As #n Thr Glu Val Arg Ser                 245   #               250   #               255 Gly Asn Leu Thr Val Gly Ser Ala Thr Ile Ph #e Val Val Tyr Met Thr             260       #           265       #           270 Gln Leu Ile Asn Pro Ile Met Gln Leu Ser Gl #n Leu Val Ala His Met         275           #       280           #       285 Gly Met Leu Asn Gly Gly Val Glu Arg Leu Le #u Glu Tyr Asn Gln Ala     290               #   295               #   300 Ile Pro Glu Lys Asn Gly Ile Lys Lys Ile As #p Glu Ile Ile Asn Ile 305                 3 #10                 3 #15                 3 #20 Ala Phe Asp Asn Val Ser Phe Ala Tyr Asp As #n Gln Glu Asn Ile Ile                 325   #               330   #               335 Glu Asn Val Asn Leu Thr Phe Gln Lys Gly Th #r Tyr Ile Ser Ile Val             340       #           345       #           350 Gly Glu Ser Gly Val Gly Lys Ser Thr Leu Le #u Asp Leu Leu Glu His         355           #       360           #       365 Asn Tyr Val Pro Ser Lys Gly Arg Ile Leu Il #e Asn Gly Ile Asp Leu     370               #   375               #   380 Glu Glu Leu Asn Ile Lys Thr Leu Arg Asn Ly #s Ile Ser Tyr Val Ser 385                 3 #90                 3 #95                 4 #00 Gln Glu Pro Thr Ile Leu Ser Gly Thr Ile Ar #g Glu Leu Leu Asp Phe                 405   #               410   #               415 Asn Gln Gln Gln His Thr Glu Thr Ser Leu Tr #p Asn Val Leu Asp Thr             420       #           425       #           430 Val Glu Leu Ser Glu Leu Ile Arg Asn Leu Pr #o Ala Lys Leu Asp Ser         435           #       440           #       445 Lys Val Asp Glu Tyr Gly Gly Asn Leu Ser Gl #y Gly Gln Met Gln Arg     450               #   455               #   460 Ile Ser Leu Ala Arg Gly Leu Leu Lys Ala Gl #y Asp Val Leu Leu Leu 465                 4 #70                 4 #75                 4 #80 Asp Glu Ser Phe Ala Asn Ile Asp Glu Glu Th #r Cys Leu Lys Ile Lys                 485   #               490   #               495 Leu Lys Ile Ala Ala Tyr Ala Glu Ser His Ly #s Gln Ile Val Ile Glu             500       #           505       #           510 Val Ile His Asn Leu Asn Arg Ile Thr Pro Se #r Ser Ile Val Tyr Arg         515           #       520           #       525 Leu Ala Asp Lys Lys Leu Glu Ile Leu Arg Se #r Gly Phe     530               #   535               #   540 <210> SEQ ID NO 12 <211> LENGTH: 233 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 12 Met Asp Tyr Met Leu Glu Thr Lys Asn Leu Th #r Lys Gln Phe Gly Lys 1               5    #                10   #                15 Gln Thr Ala Val Asn Gln Leu Asn Leu Lys Va #l Glu Arg His Ser Ile             20       #            25       #            30 Tyr Gly Leu Leu Gly Pro Asn Gly Ser Gly Ly #s Ser Thr Thr Leu Lys         35           #        40           #        45 Met Ile Thr Gly Met Leu Arg Lys Thr Ser Gl #y His Ile Leu Ile Asp     50               #    55               #    60 Gly His Asp Trp Ser Arg Lys Asp Leu Glu As #n Ile Gly Ala Leu Ile 65                   #70                   #75                   #80 Glu Ser Pro Pro Leu Tyr Glu Asn Leu Thr Al #a Arg Glu Asn Leu Lys                 85   #                90   #                95 Val Arg Thr Leu Met Leu Gly Leu Pro Asp Se #r Arg Ile Asp Glu Val             100       #           105       #           110 Leu Lys Ile Val Asp Leu Thr Asn Thr Gly Ly #s Lys Arg Ala Gly Gln         115           #       120           #       125 Phe Ser Met Gly Met Lys Gln Arg Leu Gly Il #e Ala Ile Ala Leu Leu     130               #   135               #   140 Asn Ser Pro Gln Leu Leu Ile Leu Asp Glu Pr #o Thr Asn Gly Leu Asp 145                 1 #50                 1 #55                 1 #60 Pro Ile Gly Ile Gln Glu Leu Arg Asn Leu Il #e Arg Ser Phe Pro Thr                 165   #               170   #               175 Gln Gly Ile Thr Val Ile Ile Ser Ser His Il #e Leu Ser Glu Ile Gln             180       #           185       #           190 Met Thr Ala Asp His Ile Gly Ile Ile Ala As #n Gly Val Leu Gly Tyr         195           #       200           #       205 Gln Asp Arg Ile His Gln Asp Glu Asp Leu Gl #u Lys Leu Phe Thr Asp     210               #   215               #   220 Val Val Met Arg Tyr Arg Gly Gly Glu 225                 2 #30 <210> SEQ ID NO 13 <211> LENGTH: 251 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 13 Met Leu Gly Met Phe Gln Ala Glu Arg Leu Ly #s Leu Lys Arg Ser Met 1               5    #                10   #                15 Ala Lys Lys Leu Leu Val Phe Ala Pro Ile Il #e Ala Ile Leu Tyr Gly             20       #            25       #            30 Phe Ile Ala Pro Val Gly Tyr Leu Val Asn As #n Ala Tyr Asn Trp Trp         35           #        40           #        45 Tyr Val Met Ile Phe Pro Gly Leu Leu Thr Le #u Phe Ala Ala Leu Ile     50               #    55               #    60 Asn Thr Tyr Glu Glu Lys Lys Leu His Tyr Ar #g Ala Val Phe Pro Leu 65                   #70                   #75                   #80 Pro Ile Ser Leu Arg Lys Phe Trp Phe Glu Ly #s Ile Phe Ile Thr Val                 85   #                90   #                95 Tyr Tyr Leu Asn Phe Ser Asn Gly Val Leu Tr #p Ile Ile Thr Val Leu             100       #           105       #           110 Leu Asn Thr Phe Ile Leu Pro Asn Tyr Gly Ly #s Asp Tyr Thr Tyr Thr         115           #       120           #       125 Val Gly Glu Leu Ala Leu Ala Ser Leu Val Il #e Ile Val Thr Thr Leu     130               #   135               #   140 Trp Gln Ile Pro Phe Cys Leu Trp Leu Thr Ly #s Arg Ile Gly Phe Thr 145                 1 #50                 1 #55                 1 #60 Ile Thr Leu Ile Ile Asn Leu Met Ser Asn Ph #e Ile Leu Gly Val Val                 165   #               170   #               175 Phe Ala Thr Thr Ser Cys Trp Trp Leu Cys Pr #o Tyr Ser Trp Gly Ile             180       #           185       #           190 Arg Leu Met Val Pro Ile Leu Lys Ile Leu Pr #o Ser Gly Leu Lys Ala         195           #       200           #       205 Gly Ile Ala Gly Ala Pro Ser Leu Pro Thr Se #r Phe Trp Ser Ile Val     210               #   215               #   220 Ile Ser Leu Cys Leu Ala Val Ile Leu Phe Va #l Ser Leu Thr Val Leu 225                 2 #30                 2 #35                 2 #40 Ser Ala Ser Trp Phe Glu Lys Gln Glu Val Ly #s                 245   #               250 <210> SEQ ID NO 14 <211> LENGTH: 246 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 14 Met Ile Asp Leu Leu Lys Ala Glu Asn Val Ly #s Tyr Arg His Thr Phe 1               5    #                10   #                15 Leu Pro Trp Leu His Leu Ile Leu Pro Val Th #r Thr Ala Ile Val Val             20       #            25       #            30 Ile Val Tyr Gly Leu Met Thr Pro Thr His Se #r Trp Ala Asp Ile Thr         35           #        40           #        45 Gly Gly Tyr Leu Glu Leu Leu Gly Ile Ser Ph #e Pro Ile Val Ile Ala     50               #    55               #    60 Val Ile Cys Gly Lys Ser Val Gly Leu Glu Va #l Glu Ala Gly Gln Phe 65                   #70                   #75                   #80 Gln Val Met Leu Ala Ile Lys Gln Arg Asn Le #u Ile Phe Cys Ile Lys                 85   #                90   #                95 Leu Leu Asn Leu Leu Ile Leu Glu Leu Phe Se #r Thr Leu Leu Ala Ile             100       #           105       #           110 Gly Ile Tyr Gly Leu Ile Tyr Gln Leu Ser As #n Lys His Leu Ile Phe         115           #       120           #       125 Tyr Gly Tyr Ala Val Ile Leu Leu Thr Ala Se #r Met Leu Ile Leu Tyr     130               #   135               #   140 Leu Ile His Leu Val Val Val Phe Leu Phe Gl #y Asn Ser Ala Asn Ile 145                 1 #50                 1 #55                 1 #60 Gly Leu Gly Ile Ala Glu Ser Leu Leu Ser Al #a Leu Leu Leu Thr Gly                 165   #               170   #               175 Leu Gly Asp Gly Ile Trp Gln Phe Ile Pro Cy #s Ala Trp Gly Thr Arg             180       #           185       #           190 Leu Met Gly Thr Leu Ile Asn Leu Trp Tyr Ty #r Ser Gly His Ser Leu         195           #       200           #       205 Phe Phe Lys Gln Gln Leu Leu Ile Trp Leu Gl #u Val Ala Val Pro Leu     210               #   215               #   220 Thr Leu Met Ala Leu Ile Leu Ser Ile Ile Tr #p Phe Asp Arg Trp Gln 225                 2 #30                 2 #35                 2 #40 Gly Arg Ser Ser Asp Glu                 245 <210> SEQ ID NO 15 <211> LENGTH: 246 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 15 Met Thr Tyr Ile Gly Val Ser His Leu Lys Ly #s Val Tyr Lys Thr Gln 1               5    #                10   #                15 Glu Gly Leu Thr Asn Glu Ala Leu Lys Asp Il #e Thr Phe Ser Val Gln             20       #            25       #            30 Glu Gly Glu Phe Ile Ala Ile Met Gly Glu Se #r Gly Ser Gly Lys Ser         35           #        40           #        45 Thr Leu Leu Asn Ile Leu Ala Cys Met Asp Ty #r Pro Ser Ser Gly His     50               #    55               #    60 Ile Ile Phe Asn Asn Tyr Gln Leu Glu Lys Va #l Lys Asp Glu Glu Ala 65                   #70                   #75                   #80 Ala Val Phe Arg Ser Arg His Ile Gly Phe Il #e Phe Gln Asn Phe Asn                 85   #                90   #                95 Leu Leu Asn Ile Phe Asn Asn Lys Asp Asn Le #u Leu Ile Pro Val Ile             100       #           105       #           110 Ile Ser Gly Ser Lys Val Asn Ser Tyr Glu Ly #s Arg Leu Arg Asp Leu         115           #       120           #       125 Ala Ala Val Val Gly Ile Glu Ser Leu Leu Se #r Lys Tyr Pro Tyr Glu     130               #   135               #   140 Leu Ser Gly Gly Gln Gln Gln Arg Leu Ala Il #e Ala Arg Ala Leu Ile 145                 1 #50                 1 #55                 1 #60 Met Asn Pro Asp Leu Ile Leu Ala Asp Glu Pr #o Thr Gly Gln Leu Asp                 165   #               170   #               175 Ser Lys Thr Ser Gln Arg Ile Leu Asn Leu Le #u Ser Asn Ile Asn Ala             180       #           185       #           190 Lys Arg Lys Thr Ile Leu Met Val Thr His Se #r Pro Lys Ala Ala Ser         195           #       200           #       205 Tyr Ala Asn Arg Val Leu Phe Ile Lys Asp Gl #y Val Ile Phe Asn Gln     210               #   215               #   220 Leu Val Arg Gly Cys Lys Ser Arg Glu Gly Ph #e Leu Asp Gln Ile Ile 225                 2 #30                 2 #35                 2 #40 Met Ala Gln Ala Ser Leu                 245 <210> SEQ ID NO 16 <211> LENGTH: 640 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 16 Met Phe Leu Pro Lys Ile Ser Phe His Asn Le #u Ile Val Asn Lys Ser 1               5    #                10   #                15 Leu Thr Leu Pro Tyr Phe Ala Ile Met Thr Il #e Phe Ser Gly Phe Asn             20       #            25       #            30 Tyr Val Leu Ile Asn Phe Leu Thr Asn Pro Se #r Phe Tyr Asn Ile Pro         35           #        40           #        45 Thr Ala Arg Ile Leu Ile Asp Ile Leu Ile Ph #e Gly Phe Ile Leu Ile     50               #    55               #    60 Ser Leu Leu Met Leu Leu Tyr Gly Arg Tyr Al #a Asn Arg Phe Ile Ser 65                   #70                   #75                   #80 Asp Glu Arg Asn Ser Asn Met Gly Ile Phe Le #u Met Leu Gly Met Gly                 85   #                90   #                95 Lys Lys Gln Leu Leu Lys Ile Ile Tyr Leu Gl #u Lys Leu Tyr Leu Phe             100       #           105       #           110 Thr Gly Thr Phe Phe Gly Gly Leu Ile Phe Gl #y Phe Val Tyr Ser Lys         115           #       120           #       125 Ile Phe Phe Leu Phe Ile Arg Asn Leu Ile Va #l Ile Gly Asp Val Arg     130               #   135               #   140 Glu Gln Tyr Ser Leu Thr Ala Ile Ser Trp Le #u Leu Ile Leu Thr Phe 145                 1 #50                 1 #55                 1 #60 Phe Ile Tyr Phe Ile Ile Tyr Leu Ser Glu Ty #r Arg Leu Leu Lys Arg                 165   #               170   #               175 Gln Ser Ile Thr Val Ile Phe Asn Ser Lys Al #a Lys Arg Asp Asn Pro             180       #           185       #           190 Arg Lys Thr Ser Val Phe Val Gly Leu Phe Gl #y Leu Phe Ala Leu Leu         195           #       200           #       205 Met Gly Tyr His Phe Ala Leu Thr Ser Pro As #n Val Thr Thr Ser Phe     210               #   215               #   220 Ser Arg Phe Ile Tyr Ala Ala Cys Leu Val Th #r Leu Gly Ile Phe Cys 225                 2 #30                 2 #35                 2 #40 Thr Phe Ser Ser Gly Val Ile Met Leu Leu Th #r Val Ile Lys Lys Arg                 245   #               250   #               255 Arg Ala Ile Tyr Tyr Asn Gln Arg Arg Phe Va #l Val Ile Ala Ser Leu             260       #           265       #           270 Phe His Arg Ile Arg Ser Asn Ala Leu Ser Le #u Ala Thr Ile Cys Ile         275           #       280           #       285 Phe Ser Thr Ala Thr Leu Val Ser Leu Ser Va #l Leu Ala Ser Leu Tyr     290               #   295               #   300 Leu Ala Lys Asp Asn Met Val Arg Leu Ser Se #r Pro Arg Asp Val Thr 305                 3 #10                 3 #15                 3 #20 Val Leu Ser Thr Thr Asp Ile Glu Pro Asn Le #u Met Asp Ile Ala Thr                 325   #               330   #               335 Lys Asn His Val Thr Leu Thr Asn Arg Gln As #n Leu Lys Val Ser Gln             340       #           345       #           350 Ser Val Tyr Gly Asn Ile Lys Gly Ser His Le #u Ser Val Asp Pro Asn         355           #       360           #       365 Gly Gly Met Ala Asn Asp Tyr Gln Ile Thr Va #l Ile Ser Leu Asp Ser     370               #   375               #   380 Phe Asn Ala Ser Asn Asn Thr His Tyr Arg Le #u Lys Asn His Glu Ile 385                 3 #90                 3 #95                 4 #00 Leu Thr Tyr Val Ser Asn Gly Ala Ala Ala Pr #o Ser Ser Tyr Thr Thr                 405   #               410   #               415 Asn Gly Val Lys Leu Thr Asn Val Lys Gln Il #e Lys Arg Ile Asn Phe             420       #           425       #           430 Ile Phe Ser Pro Leu Arg Ser Met Gln Pro As #n Phe Phe Ile Ile Thr         435           #       440           #       445 Asp Asn Arg Glu Ile Ile Gln Thr Ile Leu Ly #s Glu Glu Leu Thr Trp     450               #   455               #   460 Gly Thr Met Ala Gly Tyr His Val Lys Gly Ly #s Lys Met Asn Gln Lys 465                 4 #70                 4 #75                 4 #80 Asp Phe Tyr Asp Glu Leu Glu Thr Thr Asn Ph #e Arg Gln Phe Ser Ala                 485   #               490   #               495 Asn Val Val Ser Ile Arg Gln Val Lys Ser Me #t Phe Asn Ala Leu Phe             500       #           505       #           510 Gly Gly Leu Leu Phe Val Gly Ile Ile Phe Gl #y Thr Ile Phe Ala Ile         515           #       520           #       525 Leu Thr Ala Ile Thr Ile Tyr Tyr Gln Gln Le #u Ser Glu Gly Ile Arg     530               #   535               #   540 Asp Arg Asp Asp Tyr Lys Ala Met Ile Lys Le #u Gly Met Thr Asn Lys 545                 5 #50                 5 #55                 5 #60 Thr Ile Gln Asp Ser Ile Lys Val Gln Ile As #n Phe Val Phe Ile Leu                 565   #               570   #               575 Pro Ile Ala Phe Ala Leu Leu Asn Leu Ile Ph #e Ala Leu Pro Ile Leu             580       #           585       #           590 Tyr Lys Ile Met Thr Thr Phe Gly Phe Asn As #p Ala Gly Leu Phe Leu         595           #       600           #       605 Arg Ala Val Gly Thr Cys Leu Ile Val Tyr Le #u Phe Phe Tyr Trp Phe     610               #   615               #   620 Ile Cys His Cys Thr Ser Lys Leu Tyr Tyr Ar #g Leu Ile Ser Lys Lys 625                 6 #30                 6 #35                 6 #40 <210> SEQ ID NO 17 <211> LENGTH: 118 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 17 Met Arg Ile Val Ser Ser Leu Val Ser Leu Le #u Leu Thr Ile Phe Trp 1               5    #                10   #                15 Ile Phe Ala Ile Ala Phe Ile Pro Ile Gly As #p Gln Asn Ser Phe Asn             20       #            25       #            30 Lys Pro Glu Met Trp Phe Phe Val Phe Phe Al #a Ile Ile Ile Tyr Ser         35           #        40           #        45 Ile Val Ile Ile Ser Asp Tyr Tyr Leu Lys Se #r Phe Asn Leu Leu Lys     50               #    55               #    60 Val Tyr Gln Ile Leu Val Leu Phe Ile Ser Il #e Leu Cys Ala Leu Cys 65                   #70                   #75                   #80 Gly Leu Ser Leu Thr Ala Leu Gly Leu Lys Va #l Phe Thr Leu Ala Ile                 85   #                90   #                95 Gly Ile Val Ser Leu Val Asn Thr Ile Ile Ty #r Phe Phe Phe Ala Asn             100       #           105       #           110 Lys Lys Asp Asn Val Glu         115 

What is claimed is:
 1. An isolated and purified peptide sequence consisting of: the amino acid sequence as set forth in SEQ ID No: 2, or a pharmaceutically acceptable, ester, amide, and prodrug thereof.
 2. A pharmaceutical composition comprising an isolated and purified peptide having amino add sequence SEQ ID No: 2 and a pharmaceutically acceptable carrier. 